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Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase

2015-05-19T18:15:15Z

Eva Lucija Kozak: Undo revision 10575 by Eva Lucija Kozak (Talk)

==Uvod==
Lignocelulozne surovine so odpadni material v kmetijstvu in lesni industriji. Lahko se jih uporablja kot vir energije za proizvodnjo biogoriv, vendar je treba celulozo najprej sprostiti s kompleksov s hemicelulozo (ksilan) in ligninom. Povezanost polisaharidov in lignina v celičnih stenah predstavlja namreč problem pri encimski razgradnji rastlinske biomase, ker onemogoča dober dostop celulaz do celuloznih vlaken in zmanjša hidrolizo. Pri predelavi biomase je tako prva stopnja predobdelava s kislinami, bazami, vodikovim peroksidom ali s povišano temperaturo in tlakom.

==Izhodišča in cilji raziskave==
Bagasa je odpadni lignocelulozni material, ki nastane pri predelavi sladkornega trsa. Je primeren za proizvodnjo biogoriv, npr. bioetanola, ker ima visoko vsebnost polisaharidov in je dober substrat za encimsko razgradnjo. Bagaso v ta namen predobdelajo in nato encimsko razgradijo do reducirajočih sladkorjev (glukoza, ksiloza, celobioza itd.) in dalje fermentirajo in destilirajo.
Celulozo razgrajujejo s glukanazami (celulazami), dodajo pa še pomožne encime, predvsem ksilanaze, ki razgrajujejo hemicelulozo. Na ta način postane celuloza bolj dostopna za razgradnjo s celulazami, hkrati pa pridobijo več sladkorjev. Celulaze in ksilanaze imajo pri hidrolizi sinergistični učinek, ki pa je odvisen od načina predobdelave biomase.
Lili Jia in sodelavci (Bioresource Technology, 2015 [http://www.sciencedirect.com/science/article/pii/S0960852415002114]) so raziskali dva načina predobdelave bagase, in sicer s perocetno kislino (PAA) ter ionsko tekočino z 1-etil-3-metilimidazolijevim acetatom (EmimOAc). Preučili so vpliv teh dveh načinov predobdelave bagase na učinkovitost encimov in njihov sinergistični učinek. Sistematično so preučili encime endoglukanazo, Cel6A (GH6, s CBM, domeno za vezavo ogljikovih hidratov) iz ''Thermobifida fusca'', in dve endoksilanazi, XynZ-C (GH10, brez CBM) iz ''Clostridium thermocellum'' in Xyn11A (GH11, z XBM, notranjo domeno za vezavo celuloze/ksilana) iz ''Thermobifida fusca''.

==Potek dela==
Bagaso z delci do 200 μm so predobdelali s PAA in EmimOAc ter izmerili vsebnost sladkorjev s HPLC, vsebnost preostalega lignina so določili z merjenjem absorbance pri 240 nm, s SEM (vrstična elektronska mikroskopija) pa so opazovali spremembe površine bagase. Rentgensko difrakcijo so uporabili za merjenje razmerja med kristalinično in amorfno obliko celuloze (Manjša vsebnost celuloze v kristalinični obliki pomeni lažjo hidrolizo z encimi).
Sledila je encimska hidroliza pri čemer so uporabili kombinacije encimov s koncentracijo 2 mg encimov na g biomase. 100μl vzorce so analizirali po 0, 3, 6, 12, 24, 48 in 72 urah.

Sinergizem encimov so izračunali po formuli:
DS = Y(1+2) / (αY1 + βY2) oziroma DS = Y(1+2 + 3) / (αY1 + βY2 + γY3)

α, β in γ so deleži encimov, Y1, Y2, Y3, Y(1+2), Y(1+2 + 3) pomenijo pretvorbo glukana oz. ksilana z določenim encimov ali kombinacijo encimov. Nivo sinerizma (DS) večji od 1 pomeni sinergistični učinek encimov.

==Rezultati==
Različna načina predobdelave vplivata na spremembe v sestavi bagase. Bagasa, predobdelana z EmimOAc vsebuje več hemiceluloze, zlasti arabinoze; bagasa, predhodno obdelana s PAA, pa vsebuje večji delež celuloze v kristalinični obliki. Predobdelava z EmimOAc odstrani več lignina kot predobdelava s PAA.
Predobdelava vpliva tudi na strukturo bagase, po obdelavi s PAA se je površina bagase občutno povečala.

Ugotovili so, da je sinergistični učinek encimov odvisen od vsebnosti hemiceluloze, zlasti arabinana, in kristaliničnost celuloze v bagasi.
Največji sinergizem encimov pri pretvorbi glukana (nad 3,4) so opazili pri bagasi, ki so jo predhodno obdelali s PAA in je vsebovala nizke ravni arabinana. Največji sinergizem pri pretvorbi ksilana (nad 1,9) so opazili pri bagasi, ki so jo predhodno obdelali z EmimOAc in je vsebovala manj kristalizirane celuloze.
Pokazali so, da je za sinergizem pomembna tudi molekularna struktura encimov, saj je kombinacija encimov Cel6A in Xyn11A (brez CBM) privedla do višjega sinergizma pri pretvorbi glukana kot kombinacija encimov Cel6A in XynZ-C. Prisotnost CBM regije v encimu je pomembna za pretvorbo ksilana, ker izboljša interakcijo encima s substratom.

==Zaključek==
Bagasa predstavlja potencialno dober vir za proizvodnjo bioetanola z encimsko razgradnjo. Zaenkrat se jo kot vir energije bolj izkorišča z zažiganjem za proizvajanje toplote in elektrike, uporablja se jo tudi v proizvodnji papirja in kartona.

Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase

2015-05-19T18:13:50Z

Eva Lucija Kozak:

'''Uvod'''
Lignocelulozne surovine so odpadni material v kmetijstvu in lesni industriji. Lahko se jih uporablja kot vir energije za proizvodnjo biogoriv, vendar je treba celulozo najprej sprostiti s kompleksov s hemicelulozo (ksilan) in ligninom. Povezanost polisaharidov in lignina v celičnih stenah predstavlja namreč problem pri encimski razgradnji rastlinske biomase, ker onemogoča dober dostop celulaz do celuloznih vlaken in zmanjša hidrolizo. Pri predelavi biomase je tako prva stopnja predobdelava s kislinami, bazami, vodikovim peroksidom ali s povišano temperaturo in tlakom.

'''Izhodišča in cilji raziskave'''
Bagasa je odpadni lignocelulozni material, ki nastane pri predelavi sladkornega trsa. Je primeren za proizvodnjo biogoriv, npr. bioetanola, ker ima visoko vsebnost polisaharidov in je dober substrat za encimsko razgradnjo. Bagaso v ta namen predobdelajo in nato encimsko razgradijo do reducirajočih sladkorjev (glukoza, ksiloza, celobioza itd.) in dalje fermentirajo in destilirajo.
Celulozo razgrajujejo s glukanazami (celulazami), dodajo pa še pomožne encime, predvsem ksilanaze, ki razgrajujejo hemicelulozo. Na ta način postane celuloza bolj dostopna za razgradnjo s celulazami, hkrati pa pridobijo več sladkorjev. Celulaze in ksilanaze imajo pri hidrolizi sinergistični učinek, ki pa je odvisen od načina predobdelave biomase.
Lili Jia in sodelavci (Bioresource Technology, 2015 [http://www.sciencedirect.com/science/article/pii/S0960852415002114]) so raziskali dva načina predobdelave bagase, in sicer s perocetno kislino (PAA) ter ionsko tekočino z 1-etil-3-metilimidazolijevim acetatom (EmimOAc). Preučili so vpliv teh dveh načinov predobdelave bagase na učinkovitost encimov in njihov sinergistični učinek. Sistematično so preučili encime endoglukanazo, Cel6A (GH6, s CBM, domeno za vezavo ogljikovih hidratov) iz ''Thermobifida fusca'', in dve endoksilanazi, XynZ-C (GH10, brez CBM) iz ''Clostridium thermocellum'' in Xyn11A (GH11, z XBM, notranjo domeno za vezavo celuloze/ksilana) iz ''Thermobifida fusca''.

'''Potek dela'''
Bagaso z delci do 200 μm so predobdelali s PAA in EmimOAc ter izmerili vsebnost sladkorjev s HPLC, vsebnost preostalega lignina so določili z merjenjem absorbance pri 240 nm, s SEM (vrstična elektronska mikroskopija) pa so opazovali spremembe površine bagase. Rentgensko difrakcijo so uporabili za merjenje razmerja med kristalinično in amorfno obliko celuloze (Manjša vsebnost celuloze v kristalinični obliki pomeni lažjo hidrolizo z encimi).
Sledila je encimska hidroliza pri čemer so uporabili kombinacije encimov s koncentracijo 2 mg encimov na g biomase. 100μl vzorce so analizirali po 0, 3, 6, 12, 24, 48 in 72 urah.

Sinergizem encimov so izračunali po formuli:
DS = Y(1+2) / (αY1 + βY2) oziroma DS = Y(1+2 + 3) / (αY1 + βY2 + γY3)

α, β in γ so deleži encimov, Y1, Y2, Y3, Y(1+2), Y(1+2 + 3) pomenijo pretvorbo glukana oz. ksilana z določenim encimov ali kombinacijo encimov. Nivo sinerizma (DS) večji od 1 pomeni sinergistični učinek encimov.

'''Rezultati'''
Različna načina predobdelave vplivata na spremembe v sestavi bagase. Bagasa, predobdelana z EmimOAc vsebuje več hemiceluloze, zlasti arabinoze; bagasa, predhodno obdelana s PAA, pa vsebuje večji delež celuloze v kristalinični obliki. Predobdelava z EmimOAc odstrani več lignina kot predobdelava s PAA.
Predobdelava vpliva tudi na strukturo bagase, po obdelavi s PAA se je površina bagase občutno povečala.

Ugotovili so, da je sinergistični učinek encimov odvisen od vsebnosti hemiceluloze, zlasti arabinana, in kristaliničnost celuloze v bagasi.
Največji sinergizem encimov pri pretvorbi glukana (nad 3,4) so opazili pri bagasi, ki so jo predhodno obdelali s PAA in je vsebovala nizke ravni arabinana. Največji sinergizem pri pretvorbi ksilana (nad 1,9) so opazili pri bagasi, ki so jo predhodno obdelali z EmimOAc in je vsebovala manj kristalizirane celuloze.
Pokazali so, da je za sinergizem pomembna tudi molekularna struktura encimov, saj je kombinacija encimov Cel6A in Xyn11A (brez CBM) privedla do višjega sinergizma pri pretvorbi glukana kot kombinacija encimov Cel6A in XynZ-C. Prisotnost CBM regije v encimu je pomembna za pretvorbo ksilana, ker izboljša interakcijo encima s substratom.

'''Zaključek'''
Bagasa predstavlja potencialno dober vir za proizvodnjo bioetanola z encimsko razgradnjo. Zaenkrat se jo kot vir energije bolj izkorišča z zažiganjem za proizvajanje toplote in elektrike, uporablja se jo tudi v proizvodnji papirja in kartona.

Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase

2015-05-18T21:38:58Z

Eva Lucija Kozak:

Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase

2015-05-18T21:25:33Z

Eva Lucija Kozak:

==Uvod==
Lignocelulozne surovine so odpadni material v kmetijstvu in lesni industriji. Lahko se jih uporablja kot vir energije za proizvodnjo biogoriv, vendar je treba celulozo najprej sprostiti s kompleksov s hemicelulozo (ksilan) in ligninom. Povezanost polisaharidov in lignina v celičnih stenah predstavlja namreč problem pri encimski razgradnji rastlinske biomase, ker onemogoča dober dostop celulaz do celuloznih vlaken in zmanjša hidrolizo. Pri predelavi biomase je tako prva stopnja predobdelava s kislinami, bazami, vodikovim peroksidom ali s povišano temperaturo in tlakom.

==Izhodišča in cilji raziskave==
Bagasa je odpadni lignocelulozni material, ki nastane pri predelavi sladkornega trsa. Je primeren za proizvodnjo biogoriv, npr. bioetanola, ker ima visoko vsebnost polisaharidov in je dober substrat za encimsko razgradnjo. Bagaso v ta namen predobdelajo in nato encimsko razgradijo do reducirajočih sladkorjev (glukoza, ksiloza, celobioza itd.) in dalje fermentirajo in destilirajo.
Celulozo razgrajujejo s glukanazami (celulazami), dodajo pa še pomožne encime, predvsem ksilanaze, ki razgrajujejo hemicelulozo. Na ta način postane celuloza bolj dostopna za razgradnjo s celulazami, hkrati pa pridobijo več sladkorjev. Celulaze in ksilanaze imajo pri hidrolizi sinergistični učinek, ki pa je odvisen od načina predobdelave biomase.
Lili Jia in sodelavci so raziskali dva načina predobdelave bagase, in sicer s perocetno kislino (PAA) ter ionsko tekočino z 1-etil-3-metilimidazolijevim acetatom (EmimOAc). Preučili so vpliv teh dveh načinov predobdelave bagase na učinkovitost encimov in njihov sinergistični učinek. Sistematično so preučili encime endoglukanazo, Cel6A (GH6, s CBM, domeno za vezavo ogljikovih hidratov) iz ''Thermobifida fusca'', in dve endoksilanazi, XynZ-C (GH10, brez CBM) iz ''Clostridium thermocellum'' in Xyn11A (GH11, z XBM, notranjo domeno za vezavo celuloze/ksilana) iz ''Thermobifida fusca''.

==Potek dela==
Bagaso z delci do 200 μm so predobdelali s PAA in EmimOAc ter izmerili vsebnost sladkorjev s HPLC, vsebnost preostalega lignina so določili z merjenjem absorbance pri 240 nm, s SEM (vrstična elektronska mikroskopija) pa so opazovali spremembe površine bagase. Rentgensko difrakcijo so uporabili za merjenje razmerja med kristalinično in amorfno obliko celuloze (Manjša vsebnost celuloze v kristalinični obliki pomeni lažjo hidrolizo z encimi).
Sledila je encimska hidroliza pri čemer so uporabili kombinacije encimov s koncentracijo 2 mg encimov na g biomase. 100μl vzorce so analizirali po 0, 3, 6, 12, 24, 48 in 72 urah.

Sinergizem encimov so izračunali po formuli:
DS = Y(1+2) / (αY1 + βY2) oziroma DS = Y(1+2 + 3) / (αY1 + βY2 + γY3)

α, β in γ so deleži encimov, Y1, Y2, Y3, Y(1+2), Y(1+2 + 3) pomenijo pretvorbo glukana oz. ksilana z določenim encimov ali kombinacijo encimov. Nivo sinerizma (DS) večji od 1 pomeni sinergistični učinek encimov.

==Rezultati==
Različna načina predobdelave vplivata na spremembe v sestavi bagase. Bagasa, predobdelana z EmimOAc vsebuje več hemiceluloze, zlasti arabinoze; bagasa, predhodno obdelana s PAA, pa vsebuje večji delež celuloze v kristalinični obliki. Predobdelava z EmimOAc odstrani več lignina kot predobdelava s PAA.
Predobdelava vpliva tudi na strukturo bagase, po obdelavi s PAA se je površina bagase občutno povečala.

Ugotovili so, da je sinergistični učinek encimov odvisen od vsebnosti hemiceluloze, zlasti arabinana, in kristaliničnost celuloze v bagasi.
Največji sinergizem encimov pri pretvorbi glukana (nad 3,4) so opazili pri bagasi, ki so jo predhodno obdelali s PAA in je vsebovala nizke ravni arabinana (0,9%). Največji sinergizem pri pretvorbi ksilana (nad 1,9) so opazili pri bagasi, ki so jo predhodno obdelali z EmimOAc in je vsebovala manj kristalizirane celuloze.
Pokazali so, da je za sinergizem pomembna tudi molekularna struktura encimov, saj je kombinacija encimov Cel6A in Xyn11A (brez CBM) privedla do višjega sinergizma pri pretvorbi glukana kot kombinacija encimov Cel6A in XynZ-C. Prisotnost CBM regije v encimu je pomembna za pretvorbo ksilana, ker izboljša interakcijo encima s substratom.

==Zaključek==
Bagasa predstavlja potencialno dober vir za proizvodnjo bioetanola z encimsko razgradnjo. Zaenkrat se jo kot vir energije bolj izkorišča z zažiganjem za proizvajanje toplote in elektrike, uporablja se jo tudi v proizvodnji papirja in kartona.

Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase

2015-05-18T21:15:36Z

Eva Lucija Kozak:

==Uvod==
Lignocelulozne surovine so odpadni material v kmetijstvu in lesni industriji. Lahko se jih uporablja kot vir energije za proizvodnjo biogoriv, vendar je treba celulozo najprej sprostiti s kompleksov s hemicelulozo (ksilan) in ligninom. Povezanost polisaharidov in lignina v celičnih stenah predstavlja namreč problem pri encimski razgradnji rastlinske biomase, ker onemogoča dober dostop celulaz do celuloznih vlaken in zmanjša hidrolizo. Pri predelavi biomase je tako prva stopnja predobdelava s kislinami, bazami, vodikovim peroksidom ali s povišano temperaturo in tlakom.

==Izhodišča in cilji raziskave==
Bagasa je odpadni lignocelulozni material, ki nastane pri predelavi sladkornega trsa. Je primeren za proizvodnjo biogoriv, npr. bioetanola, ker ima visoko vsebnost polisaharidov in je dober substrat za encimsko razgradnjo. Bagaso v ta namen predobdelajo in nato encimsko razgradijo do reducirajočih sladkorjev (glukoza, ksiloza, celobioza itd.) in dalje fermentirajo in destilirajo.
Celulozo razgrajujejo s glukanazami (celulazami), dodajo pa še pomožne encime, predvsem ksilanaze, ki razgrajujejo hemicelulozo. Na ta način postane celuloza bolj dostopna za razgradnjo s celulazami, hkrati pa pridobijo več sladkorjev. Celulaze in ksilanaze imajo pri hidrolizi sinergistični učinek, ki pa je odvisen od načina predobdelave biomase.
Lili Jia in sodelavci so raziskali dva načina predobdelave bagase, in sicer s perocetno kislino (PAA) ter ionsko tekočino z 1-etil-3-metilimidazolijevim acetatom (EmimOAc). Preučili so vpliv the dveh načinov predobdelave bagase na učikovitost encimov in njihov sinergistični učinek. Sistematično so preučili encime endoglukanazo, Cel6A (GH6, s CBM, domeno za vezavo ogljikovih hidratov) iz Thermobifida fusca, in dve endoksilanazi, XynZ-C (GH10, brez CBM) iz Clostridium thermocellum in Xyn11A (GH11, z XBM, notranjo domeno za vezavo celuloze/ksilana) iz Thermobifida fusca.

==Potek dela==
Bagaso z delci do 200 μm so predobdelali s PAA in EmimOAc ter izmerili vsebnost sladkorjev s HPLC, vsebnost preostalega lignina so določili z merjenjem absorbance pri 240 nm, s SEM (vrstična elektronska mikroskopija) pa so opazovali spremembe površine bagase. Rentgensko difrakcijo so uporabili za merjenje razmerja med kristalinično in amorfno obliko celuloze (Manjša vsebnost celuloze v kristalinični obliki pomeni lažjo hidrolizo z encimi).
Sledila je encimska hidroliza pri čemer so uporabili kombinacije encimov s koncentracijo 2 mg encimov na g biomase. 100μl vzorce so analizirali po 0, 3, 6, 12, 24, 48 in 72 urah.

Sinergizem encimov so izračunali po formuli:
DS = Y(1+2) / (αY1 + βY2) oziroma DS = Y(1+2 + 3) / (αY1 + βY2 + γY3)

α, β in γ so deleži encimov, Y1, Y2, Y3, Y(1+2), Y(1+2 + 3) pomenijo pretvorbo glukana oz. ksilana z določenim encimov ali kombinacijo encimov. Nivo sinerizma (DS) večji od 1 pomeni sinergistični učinek encimov.

==Rezultati==
Različna načina predobdelave vplivata na spremembe v sestavi bagase. Bagasa, predobdelana z EmimOAc vsebuje več hemiceluloze, zlasti arabinoze; bagasa, predhodno obdelana s PAA, pa vsebuje večji delež celuloze v kristalinični obliki. Predobdelava z EmimOAc odstrani več lignina kot predobdelava s PAA.
Predobdelava vpliva tudi na strukturo bagase, po tretiranju s PAA se je površina bagase občutno povčala.

Ugotovili so, da je sinergistični učinek encimov odvisen od vsebnosti hemiceluloze, zlasti arabinana, in kristaliničnost celuloze v bagasi.
Največji sinergizem encimov pri pretvorbi glukana (nad 3,4) so opazili pri bagasi, ki so jo predhodno obdelali s PAA in je vsebovala nizke ravni arabinana (0,9%). Največji sinergizem pri pretvorbi ksilana (nad 1,9) so opazili pri bagasi, ki so jo predhodno obdelali z EmimOAc in je vsebovala manj kristalizirane celuloze.
Pokazali so, da je za sinergizem pomembna tudi molekularna struktura encimov, saj je kombinacija encimov Cel6A in Xyn11A (brez CBM) privedla do višjega sinergizma pri pretvorbi glukana kot kombinacija encimov Cel6A in XynZ-C. Prisotnost CBM regije v encimu je pomembna za pretvorbo ksilana, ker izboljša interakcijo encima s substratom.

==Zaključek==
Bagasa predstavlja potencilno dober vir za proizvodnjo bioetanola z encimsko razgradnjo. Zaenkrat se jo kot vir energije bolj izkorišča z zažiganjem za proizvajanje toplote in elektrike, uporablja se jo tudi v proizvodnji papirja in kartona.

Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase

2015-05-17T17:22:20Z

Eva Lucija Kozak: New page: Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase

Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase

MBT seminarji 2015

2015-05-17T17:21:43Z

Eva Lucija Kozak:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline''' 
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# [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)
# Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015

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# Functional mutations in and characterization of VHH against ''Helicobacter pylori'' urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). [[Funkcionalne mutacije in karakterizacija VHH proti ureazi Helicobacter pylori]]. Marko Radojković, 7. maja 2015

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# Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) [[Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena.]] Daša Pavc, 8. maja 2015

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# Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) [[Integracija genov za sintezo hialuronske kisline v kromosom bakterije Lactococcus lactis izboljša sintezo visokomolekularne hialuronske kisline]] Maja Grdadolnik, 15. maja 2015

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# Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline. Jure Zabret, 29. maja 2015

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# Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production; http://www.sciencedirect.com/science/article/pii/S0960148114007022. Jana Verbančič, 5. junija 2015

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# Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka. Luka Smole, 11. junija 2015

Creation of a bacterial cell controlled by a chemically synthesized genome

2015-01-05T20:07:04Z

Eva Lucija Kozak:

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article '''[http://www.sciencemag.org/content/329/5987/52.full Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56]'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter [https://www.youtube.com/watch?v=QHIocNOHd7A (see a video)] (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretched the bounds of our common conception of (natural) life.

Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.
[http://www.nature.com/nbt/journal/v28/n7/fig_tab/nbt0710-687_F1.html (see a sheme)] (Itaya, 2010)

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the [http://www.jcvi.org/cms/home/ J. Craig Venter Institute] which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

=== BACKGROUND RESEARCH ===

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and then join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative of ''M. mycoides'' (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

=== THE SEQUENCE ===

When new sequences of genomes of different organisms are determined the information is put in a genetic bank: [http://www.ncbi.nlm.nih.gov/genbank/ GenBank]. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''subspecies ''capri''. The genome of ''Mycoplasma myocides'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010) [http://www.sciencemag.org/content/329/5987/52/F1.expansion.html (Fig. 1)]

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. (Gibson, ''et al.'' 2010) Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. The overlapping cassettes were joined by homologous recombination (see an [http://nar.oxfordjournals.org/content/31/15/4373/F1.large.jpg image]).

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1μg of each assembly per 400ml of yeast culture. 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell.
The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connected the DNA bases comprising the donor genome. Consequently, the donor genome was no longer repelled by the recipient cell membranes. The PEG changed the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contained both genomes but once this heterodiploid cell was returned to growth conditions, it divided with one genome ending up in each daughter cell and the selection was made by tetracycline. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the experiment was successful because only genome left in the cell was the one of ''M. mycoides'' which was transplanted there. (Sleator, 2010)

=== RESULTS ===

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most ''Mycoplasma''. [http://www.sciencemag.org/content/329/5987/52/F5.expansion.html (Fig. 5A)] (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

=== BLUE COLONIES ===

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

=== WATERMARKS ===

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

=== RESTRICTION ANALYSIS ===

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. [http://www.sciencemag.org/content/329/5987/52/F4.expansion.html (Fig. 4B)] (Gibson, ''et al.'' 2010)

=== SEQUENCING ===

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

=== AN UNWELCOME MUTATION ===

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in ''dnaA'', an essential gene for chromosomal replication. The deletion in ''dna''A was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

=== A PROBLEM WITH THE RESTRICTION SYSTEM ===

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

=== THE SCIENTIFIC ACHIVEMENT ===

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

=== THE RESPONSE ===

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

=== THE ARTIFICIAL LIFE ===

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. For this purpose a synthetic genome could be transplanted in a lipid vesicle. (Venter, 2014) Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
=== REFERENCES ===

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381 – 390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984 – 6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

ITAYA, M., A synthetic DNA transplant, ''Nature Biotechnology'', 2010, Vol. 28, p. 687 – 689

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632 - 638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985 - 986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79 – 89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229 - 230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687 – 701.

VENTER, C. [http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en Watch me unveil "synthetic life", 2010, 18:14 ] (available on 25. 12. 2014)

VENTER, C. [https://www.youtube.com/watch?v=Txj8UCTo9d4 Synthetic Life, 2014, 42:55] (available on 2. 1. 2015)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241 – 248

[http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ First self-replicating synthetic bacterial cell, J. Craig Venter Institute] (available on 25. 12. 2014)

[http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf Fact Sheet: JCVI’s Synthetic Genomics Research] (available on 25. 12. 2014)

[[SB students resources]]

Creation of a bacterial cell controlled by a chemically synthesized genome

2015-01-05T20:05:05Z

Eva Lucija Kozak:

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article '''[http://www.sciencemag.org/content/329/5987/52.full Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56]'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter [https://www.youtube.com/watch?v=QHIocNOHd7A (see a video)] (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretched the bounds of our common conception of (natural) life.

Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.
[http://www.nature.com/nbt/journal/v28/n7/fig_tab/nbt0710-687_F1.html (see a sheme)] (Itaya, 2010)

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the [http://www.jcvi.org/cms/home/ J. Craig Venter Institute] which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

=== BACKGROUND RESEARCH ===

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and then join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative of ''M. mycoides'' (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

=== THE SEQUENCE ===

When new sequences of genomes of different organisms are determined the information is put in a genetic bank: [http://www.ncbi.nlm.nih.gov/genbank/ GenBank]. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''subspecies ''capri''. The genome of ''Mycoplasma myocides'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010) [http://www.sciencemag.org/content/329/5987/52/F1.expansion.html (Fig. 1)]

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. (Gibson, ''et al.'' 2010) Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. The overlapping cassettes were joined by homologous recombination (see an [http://nar.oxfordjournals.org/content/31/15/4373/F1.large.jpg image]).

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1μg of each assembly per 400ml of yeast culture. 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section [[WATERMARKS]]).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell.
The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connected the DNA bases comprising the donor genome. Consequently, the donor genome was no longer repelled by the recipient cell membranes. The PEG changed the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contained both genomes but once this heterodiploid cell was returned to growth conditions, it divided with one genome ending up in each daughter cell and the selection was made by tetracycline. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the experiment was successful because only genome left in the cell was the one of ''M. mycoides'' which was transplanted there. (Sleator, 2010)

=== RESULTS ===

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most ''Mycoplasma''. [http://www.sciencemag.org/content/329/5987/52/F5.expansion.html (Fig. 5A)] (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

=== BLUE COLONIES ===

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

=== WATERMARKS ===

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

=== RESTRICTION ANALYSIS ===

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. [http://www.sciencemag.org/content/329/5987/52/F4.expansion.html (Fig. 4B)] (Gibson, ''et al.'' 2010)

=== SEQUENCING ===

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

=== AN UNWELCOME MUTATION ===

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in ''dnaA'', an essential gene for chromosomal replication. The deletion in ''dna''A was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

=== A PROBLEM WITH THE RESTRICTION SYSTEM ===

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

=== THE SCIENTIFIC ACHIVEMENT ===

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

=== THE RESPONSE ===

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

=== THE ARTIFICIAL LIFE ===

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. For this purpose a synthetic genome could be transplanted in a lipid vesicle. (Venter, 2014) Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
=== REFERENCES ===

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381 – 390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984 – 6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

ITAYA, M., A synthetic DNA transplant, ''Nature Biotechnology'', 2010, Vol. 28, p. 687 – 689

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632 - 638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985 - 986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79 – 89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229 - 230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687 – 701.

VENTER, C. [http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en Watch me unveil "synthetic life", 2010, 18:14 ] (available on 25. 12. 2014)

VENTER, C. [https://www.youtube.com/watch?v=Txj8UCTo9d4 Synthetic Life, 2014, 42:55] (available on 2. 1. 2015)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241 – 248

[http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ First self-replicating synthetic bacterial cell, J. Craig Venter Institute] (available on 25. 12. 2014)

[http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf Fact Sheet: JCVI’s Synthetic Genomics Research] (available on 25. 12. 2014)

[[SB students resources]]

Creation of a bacterial cell controlled by a chemically synthesized genome

2015-01-05T18:53:09Z

Eva Lucija Kozak: /* How did they synthesize it? */

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article '''[http://www.sciencemag.org/content/329/5987/52.full Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56]'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter [https://www.youtube.com/watch?v=QHIocNOHd7A (see a video)] (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.

Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.
[http://www.nature.com/nbt/journal/v28/n7/fig_tab/nbt0710-687_F1.html (see a sheme)] (Itaya, 2010)

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the [http://www.jcvi.org/cms/home/ J. Craig Venter Institute] which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

=== BACKGROUND RESEARCH ===

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

=== THE SEQUENCE ===

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: [http://www.ncbi.nlm.nih.gov/genbank/ GenBank]. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010) [http://www.sciencemag.org/content/329/5987/52/F1.expansion.html (Fig. 1)]

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. (Gibson, ''et al.'' 2010) Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. The overlapping cassettes were joined by homologous recombination (see [http://nar.oxfordjournals.org/content/31/15/4373/F1.large.jpg image]).

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture. 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

=== RESULTS ===

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. [http://www.sciencemag.org/content/329/5987/52/F5.expansion.html (Fig. 5A)] (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

=== BLUE COLONIES ===

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

=== WATERMARKS ===

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

=== RESTRICTION ANALYSIS ===

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. [http://www.sciencemag.org/content/329/5987/52/F4.expansion.html (Fig. 4B)] (Gibson, ''et al.'' 2010)

=== SEQUENCING ===

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

=== AN UNWELCOME MUTATION ===

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The deletion in ''dna''A was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

=== A PROBLEM WITH THE RESTRICTION SYSTEM ===

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

=== THE SCIENTIFIC ACHIVEMENT ===

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

=== THE RESPONSE ===

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

=== THE ARTIFICIAL LIFE ===

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. For this purpose a synthetic genome could be transplanted in a lipid vesicle. (Venter, 2014) Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
=== REFERENCES ===

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381 – 390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984 – 6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

ITAYA, M., A synthetic DNA transplant, ''Nature Biotechnology'', 2010, Vol. 28, p. 687 – 689

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632 - 638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985 - 986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79 – 89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229 - 230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687 – 701.

VENTER, C. [http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en Watch me unveil "synthetic life", 2010, 18:14 ] (available on 25. 12. 2014)

VENTER, C. [https://www.youtube.com/watch?v=Txj8UCTo9d4 Synthetic Life, 2014, 42:55] (available on 2. 1. 2015)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241 – 248

[http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ First self-replicating synthetic bacterial cell, J. Craig Venter Institute] (available on 25. 12. 2014)

[http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf Fact Sheet: JCVI’s Synthetic Genomics Research] (available on 25. 12. 2014)

[[SB students resources]]

Creation of a bacterial cell controlled by a chemically synthesized genome

2015-01-04T20:34:52Z

Eva Lucija Kozak:

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article '''[http://www.sciencemag.org/content/329/5987/52.full Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56]'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter [https://www.youtube.com/watch?v=QHIocNOHd7A (see a video)] (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.

Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.
[http://www.nature.com/nbt/journal/v28/n7/fig_tab/nbt0710-687_F1.html (see a sheme)] (Itaya, 2010)

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the [http://www.jcvi.org/cms/home/ J. Craig Venter Institute] which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

=== BACKGROUND RESEARCH ===

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

=== THE SEQUENCE ===

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: [http://www.ncbi.nlm.nih.gov/genbank/ GenBank]. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010) [http://www.sciencemag.org/content/329/5987/52/F1.expansion.html (Fig. 1)]

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. (Gibson, ''et al.'' 2010)

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture. 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

=== RESULTS ===

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. [http://www.sciencemag.org/content/329/5987/52/F5.expansion.html (Fig. 5A)] (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

=== BLUE COLONIES ===

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

=== WATERMARKS ===

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

=== RESTRICTION ANALYSIS ===

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. [http://www.sciencemag.org/content/329/5987/52/F4.expansion.html (Fig. 4B)] (Gibson, ''et al.'' 2010)

=== SEQUENCING ===

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

=== AN UNWELCOME MUTATION ===

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The deletion in ''dna''A was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

=== A PROBLEM WITH THE RESTRICTION SYSTEM ===

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

=== THE SCIENTIFIC ACHIVEMENT ===

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

=== THE RESPONSE ===

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

=== THE ARTIFICIAL LIFE ===

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. For this purpose a synthetic genome could be transplanted in a lipid vesicle. (Venter, 2014) Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
=== REFERENCES ===

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381 – 390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984 – 6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

ITAYA, M., A synthetic DNA transplant, ''Nature Biotechnology'', 2010, Vol. 28, p. 687 – 689

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632 - 638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985 - 986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79 – 89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229 - 230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687 – 701.

VENTER, C. [http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en Watch me unveil "synthetic life", 2010, 18:14 ] (available on 25. 12. 2014)

VENTER, C. [https://www.youtube.com/watch?v=Txj8UCTo9d4 Synthetic Life, 2014, 42:55] (available on 2. 1. 2015)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241 – 248

[http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ First self-replicating synthetic bacterial cell, J. Craig Venter Institute] (available on 25. 12. 2014)

[http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf Fact Sheet: JCVI’s Synthetic Genomics Research] (available on 25. 12. 2014)

[[SB students resources]]

Creation of a bacterial cell controlled by a chemically synthesized genome

2015-01-04T19:37:48Z

Eva Lucija Kozak: /* REFERENCES */

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article '''[http://www.sciencemag.org/content/329/5987/52.full Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56]'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter [https://www.youtube.com/watch?v=QHIocNOHd7A (see a video)] (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.

Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.
[http://www.nature.com/nbt/journal/v28/n7/fig_tab/nbt0710-687_F1.html (see a sheme)] (Itaya, 2010)

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the [http://www.jcvi.org/cms/home/ J. Craig Venter Institute] which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

=== BACKGROUND RESEARCH ===

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

=== THE SEQUENCE ===

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: [http://www.ncbi.nlm.nih.gov/genbank/ GenBank]. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010) [http://www.sciencemag.org/content/329/5987/52/F1.expansion.html (Fig. 1)]

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. (Gibson, ''et al.'' 2010)

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture. 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

=== RESULTS ===

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. [http://www.sciencemag.org/content/329/5987/52/F5.expansion.html (Fig. 5A)] (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

=== BLUE COLONIES ===

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

=== WATERMARKS ===

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

=== RESTRICTION ANALYSIS ===

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. [http://www.sciencemag.org/content/329/5987/52/F4.expansion.html (Fig. 4B)] (Gibson, ''et al.'' 2010)

=== SEQUENCING ===

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

=== AN UNWELCOME MUTATION ===

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The deletion in ''dna''A was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

=== A PROBLEM WITH THE RESTRICTION SYSTEM ===

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

=== THE SCIENTIFIC ACHIVEMENT ===

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

=== THE RESPONSE ===

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

=== THE ARTIFICIAL LIFE ===

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
=== REFERENCES ===

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381 – 390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984 – 6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

ITAYA, M., A synthetic DNA transplant, ''Nature Biotechnology'', 2010, Vol. 28, p. 687 – 689

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632 - 638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985 - 986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79 – 89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229 - 230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687 – 701.

VENTER, C. [http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en Watch me unveil "synthetic life", 18:14 ] (available on 25. 12. 2014)

VENTER, C. [https://www.youtube.com/watch?v=Txj8UCTo9d4 Synthetic Life, 42:55] (available on 2. 1. 2015)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241 – 248

[http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ First self-replicating synthetic bacterial cell, J. Craig Venter Institute] (available on 25. 12. 2014)

[http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf Fact Sheet: JCVI’s Synthetic Genomics Research] (available on 25. 12. 2014)

[[SB students resources]]

SB students resources

2015-01-04T19:30:55Z

Eva Lucija Kozak:

===Introduction to our students resources in Synthetic Biology===
(Marko Dolinar)

Synthetic biology made a vast progress in good 10 years since it established itself as an interdisciplinary field of research on the interface of molecular biology and engineering. University of Ljubljana Faculty of Chemistry and Chemical Technology has introduced a Synthetic Biology course as a part od Biochemistry MSc programme only in 2013/14. This is relatively late, considering a great success of Slovenian students at iGEM competitions since their first attendance in 2006. On the other hand, the field is still in its first stages if development and a complete textbook for a MSc level course is still missing. This is the reason why our students collaborated on the preparation of a Synthetic Biology textbook with the working title Synthetic Biology - A Students Textbook. It exists as a draft that is not publicly available and is actually part 1 of a (to be) 2-volumes title. Part I is subtitled Engineering Biology, while Part II (that currently doesn't exisist yet) will be subtitled Synthetic Biology Applications.

As in all highly competitive fields of science and technology, students should be following recent progress by reading articles in high quality journals. However, this is often a very difficult task, especially at the BSc level. Specificities of the scientific and technical language, push of publishers towards very short methodological chapters and limited knowledge studens might have about advanced techniques make understanding papers a very challenging task. Therefore, I decided to face MSc students with the challenge to explain selected SB articles in a manner that would make the content of these articles understandable to BSc level students and non-experts.

In 2014/15, seminars in Synthetic Biology include explanations and presentations of some of the top-cited articles from the field of Synthetic Biology. I compiled a list of 95 articles published between 2000 and 2014 having the highest number of citations according to the Web of Science database. The list ends with the paper just exceeding the 100 citations limit. Not included in the list were reviews. With 20 students enrolled in the course, the list has been further reduced to top 40 papers in the field. Students have been asked to check for content (they further eliminated 3 papers which proved to be reviews) and availabitly (they all seemed to be available as full texts with our university subscriptions). My suggestion was to avoid selecting for presentation papers with very similar content. Especially in the field of genome editing there has been a very rapid progress in the past few years resulting in a number of highly-cited articles which could appear very similar in content for a non-specialist. From the shortlist of 37 articles, students selected a topic they believed would be most interesting or easiest to explain. Presentations Will be both written (in English, which is not the mother tongue of my students) and oral (in Slovenian, to establish and maintain Slovenian terminology in the field).

===List of articles for presentation===

This is the list of top-cited papers from the broader field of Synthetic Biology that students chose for explanation in 2014/15 (sorted by year of publication):

#[[A synthetic oscillatory network of transcriptional regulators]], Michael B. Elowitz & Stanislas Leibler, Letters to Nature, 2000 - Valter Bergant
#[[Construction of a genetic toggle switch in Escherichia coli]]. Gardner ''et al''., Nature, 2000 - Urban Bezeljak
#Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion (2001) - Andreja Bratovš
#Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template (2002) - Veronika Jarc
#[[Combinatorial synthesis of genetic networks]]. Guet C.C. ''et al'', Science, 2002 - Maja Remškar
#Engineering a mevalonate pathway in Escherichia coli for production of terpenoids (2003) - Ana Kapraljević
#Programmed population control by cell-cell communication and regulated killing (2004) - Alja Zottel
#Gene regulation at the single-cell level (2005) - Katarina Uršič
#A synthetic multicellular system for programmed pattern formation (2005) - Mitja Crček
#Long-term monitoring of bacteria undergoing programmed population control in a microchemostat (2005) - Jana Verbančič
#Tuning genetic control through promoter engineering (2005) - Špela Pohleven
#Production of the antimalarial drug precursor artemisinic acid in engineered yeast (2006) - Živa Marsetič
#An improved zinc-finger nuclease architecture for highly specific genome editing (2007) - Eva Knapič
#Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases (2008) - Tamara Marić
#Synthetic protein scaffolds provide modular control over metabolic flux (2009) - Ana Dolinar
#[[Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome]]. Gibson, D. G. ''et al.'', Science, 2010 - Eva Lucija Kozak
#A TALE nuclease architecture for efficient genome editing (2011) Jernej Mustar
#Multiplex genome engineering using CRISPR/Cas systems (2013) - Uroš Stupar
#RNA-guided human genome engineering via Cas9 (2013) - Luka Smole
#One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering (2013) - Andrej Vrankar

''Please link the title of each paper with your written seminar wiki page. Expand the citation according to the following example:
''
#Emergent bistability by a growth-modulating positive feedback circuit. Tan et al., Nature Chem. Biol., 2009

Creation of a bacterial cell controlled by a chemically synthesized genome

2015-01-04T13:51:56Z

Eva Lucija Kozak: /* AN UNWELCOME MUTATION */

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article '''[http://www.sciencemag.org/content/329/5987/52.full Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56]'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter [https://www.youtube.com/watch?v=QHIocNOHd7A (see a video)] (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.

Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.
[http://www.nature.com/nbt/journal/v28/n7/fig_tab/nbt0710-687_F1.html (see a sheme)] (Itaya, 2010)

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the [http://www.jcvi.org/cms/home/ J. Craig Venter Institute] which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

=== BACKGROUND RESEARCH ===

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

=== THE SEQUENCE ===

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: [http://www.ncbi.nlm.nih.gov/genbank/ GenBank]. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010) [http://www.sciencemag.org/content/329/5987/52/F1.expansion.html (Fig. 1)]

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. (Gibson, ''et al.'' 2010)

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture. 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

=== RESULTS ===

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. [http://www.sciencemag.org/content/329/5987/52/F5.expansion.html (Fig. 5A)] (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

=== BLUE COLONIES ===

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

=== WATERMARKS ===

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

=== RESTRICTION ANALYSIS ===

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. [http://www.sciencemag.org/content/329/5987/52/F4.expansion.html (Fig. 4B)] (Gibson, ''et al.'' 2010)

=== SEQUENCING ===

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

=== AN UNWELCOME MUTATION ===

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The deletion in ''dna''A was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

=== A PROBLEM WITH THE RESTRICTION SYSTEM ===

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

=== THE SCIENTIFIC ACHIVEMENT ===

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

=== THE RESPONSE ===

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

=== THE ARTIFICIAL LIFE ===

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
=== REFERENCES ===

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381 – 390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984 – 6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

ITAYA, M., A synthetic DNA transplant, ''Nature Biotechnology'', 2010, Vol. 28, p. 687 – 689

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632 - 638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985 - 986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79 – 89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229 - 230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687 – 701.

VENTER, C. Watch me unveil "synthetic life" 18:14
http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en (available on 25. 12. 2014)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241 – 248

[http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ First self-replicating synthetic bacterial cell, J. Craig Venter Institute] (available on 25. 12. 2014)

[http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf Fact Sheet: JCVI’s Synthetic Genomics Research] (available on 25. 12. 2014)

Creation of a bacterial cell controlled by a chemically synthesized genome

2015-01-04T13:12:46Z

Eva Lucija Kozak: /* How did they synthesize it? */

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article '''[http://www.sciencemag.org/content/329/5987/52.full Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56]'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter [https://www.youtube.com/watch?v=QHIocNOHd7A (see a video)] (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.

Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.
[http://www.nature.com/nbt/journal/v28/n7/fig_tab/nbt0710-687_F1.html (see a sheme)] (Itaya, 2010)

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the [http://www.jcvi.org/cms/home/ J. Craig Venter Institute] which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

=== BACKGROUND RESEARCH ===

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

=== THE SEQUENCE ===

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: [http://www.ncbi.nlm.nih.gov/genbank/ GenBank]. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010) [http://www.sciencemag.org/content/329/5987/52/F1.expansion.html (Fig. 1)]

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. (Gibson, ''et al.'' 2010)

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture. 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

=== RESULTS ===

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. [http://www.sciencemag.org/content/329/5987/52/F5.expansion.html (Fig. 5A)] (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

=== BLUE COLONIES ===

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

=== WATERMARKS ===

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

=== RESTRICTION ANALYSIS ===

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. [http://www.sciencemag.org/content/329/5987/52/F4.expansion.html (Fig. 4B)] (Gibson, ''et al.'' 2010)

=== SEQUENCING ===

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

=== AN UNWELCOME MUTATION ===

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The dnaA mutation was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

=== A PROBLEM WITH THE RESTRICTION SYSTEM ===

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

=== THE SCIENTIFIC ACHIVEMENT ===

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

=== THE RESPONSE ===

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

=== THE ARTIFICIAL LIFE ===

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
=== REFERENCES ===

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381 – 390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984 – 6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

ITAYA, M., A synthetic DNA transplant, ''Nature Biotechnology'', 2010, Vol. 28, p. 687 – 689

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632 - 638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985 - 986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79 – 89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229 - 230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687 – 701.

VENTER, C. Watch me unveil "synthetic life" 18:14
http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en (available on 25. 12. 2014)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241 – 248

[http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ First self-replicating synthetic bacterial cell, J. Craig Venter Institute] (available on 25. 12. 2014)

[http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf Fact Sheet: JCVI’s Synthetic Genomics Research] (available on 25. 12. 2014)

Creation of a bacterial cell controlled by a chemically synthesized genome

2015-01-04T11:38:25Z

Eva Lucija Kozak:

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article '''[http://www.sciencemag.org/content/329/5987/52.full Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56]'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter [https://www.youtube.com/watch?v=QHIocNOHd7A (see a video)] (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.

Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.
[http://www.nature.com/nbt/journal/v28/n7/fig_tab/nbt0710-687_F1.html (see a sheme)] (Itaya, 2010)

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the [http://www.jcvi.org/cms/home/ J. Craig Venter Institute] which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

=== BACKGROUND RESEARCH ===

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

=== THE SEQUENCE ===

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: [http://www.ncbi.nlm.nih.gov/genbank/ GenBank]. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010) [http://www.sciencemag.org/content/329/5987/52/F1.expansion.html (Fig. 1)]

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. (Gibson, ''et al.'' 2010)

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture (~1011 cells). 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

=== RESULTS ===

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. [http://www.sciencemag.org/content/329/5987/52/F5.expansion.html (Fig. 5A)] (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

=== BLUE COLONIES ===

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

=== WATERMARKS ===

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

=== RESTRICTION ANALYSIS ===

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. [http://www.sciencemag.org/content/329/5987/52/F4.expansion.html (Fig. 4B)] (Gibson, ''et al.'' 2010)

=== SEQUENCING ===

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

=== AN UNWELCOME MUTATION ===

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The dnaA mutation was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

=== A PROBLEM WITH THE RESTRICTION SYSTEM ===

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

=== THE SCIENTIFIC ACHIVEMENT ===

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

=== THE RESPONSE ===

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

=== THE ARTIFICIAL LIFE ===

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
=== REFERENCES ===

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381 – 390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984 – 6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

ITAYA, M., A synthetic DNA transplant, ''Nature Biotechnology'', 2010, Vol. 28, p. 687 – 689

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632 - 638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985 - 986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79 – 89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229 - 230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687 – 701.

VENTER, C. Watch me unveil "synthetic life" 18:14
http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en (available on 25. 12. 2014)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241 – 248

[http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ First self-replicating synthetic bacterial cell, J. Craig Venter Institute] (available on 25. 12. 2014)

[http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf Fact Sheet: JCVI’s Synthetic Genomics Research] (available on 25. 12. 2014)

Creation of a bacterial cell controlled by a chemically synthesized genome

2014-12-28T16:34:21Z

Eva Lucija Kozak:

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article '''Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.
Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the J. Craig Venter Institute which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

=== BACKGROUND RESEARCH ===

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

=== THE SEQUENCE ===

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: GenBank. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010)

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. (Gibson, ''et al.'' 2010)

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture (~1011 cells). 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

=== RESULTS ===

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

=== BLUE COLONIES ===

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

=== WATERMARKS ===

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

=== RESTRICTION ANALYSIS ===

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. (Gibson, ''et al.'' 2010)

=== SEQUENCING ===

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

=== AN UNWELCOME MUTATION ===

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The dnaA mutation was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

=== A PROBLEM WITH THE RESTRICTION SYSTEM ===

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

=== THE SCIENTIFIC ACHIVEMENT ===

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

=== THE RESPONSE ===

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

=== THE ARTIFICIAL LIFE ===

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
=== REFERENCES ===

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381–390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984–6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632-638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985-986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79–89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229-230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687–701.

VENTER, C. Watch me unveil "synthetic life" 18:14
http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en (available on 25. 12. 2014)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241–248

http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ (available on 25. 12. 2014)

Fact Sheet: JCVI’s Synthetic Genomics Research, http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf (available on 25. 12. 2014)

Creation of a bacterial cell controlled by a chemically synthesized genome

2014-12-28T16:29:38Z

Eva Lucija Kozak:

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists '''Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang''' and their colleagues at the Craig Venter's laboratory in 2010 and described in the article Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' '''Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56'''. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.
Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.

=== ''Mycoplasma'' ===

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

=== J. Craig Venter Institute ===

The research was done at the J. Craig Venter Institute which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

== BACKGROUND RESEARCH ==

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

== THE SEQUENCE ==

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: GenBank. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010)

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. (Gibson, ''et al.'' 2010)

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture (~1011 cells). 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

== RESULTS ==

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

== BLUE COLONIES ==

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

== WATERMARKS ==

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

== RESTRICTION ANALYSIS ==

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. (Gibson, ''et al.'' 2010)

== SEQUENCING ==

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

== AN UNWELCOME MUTATION ==

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The dnaA mutation was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

== A PROBLEM WITH THE RESTRICTION SYSTEM ==

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

== THE SCIENTIFIC ACHIVEMENT ==

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

== THE RESPONSE ==

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

== THE ARTIFICIAL LIFE ==

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
== REFERENCES ==

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381–390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984–6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632-638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985-986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79–89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229-230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687–701.

VENTER, C. Watch me unveil "synthetic life" 18:14
http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en (available on 25. 12. 2014)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241–248

http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ (available on 25. 12. 2014)

Fact Sheet: JCVI’s Synthetic Genomics Research, http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf (available on 25. 12. 2014)

Creation of a bacterial cell controlled by a chemically synthesized genome

2014-12-28T16:26:22Z

Eva Lucija Kozak:

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.
Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.

== ''Mycoplasma'' ==

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

== J. Craig Venter Institute ==

The research was done at the J. Craig Venter Institute which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

== BACKGROUND RESEARCH ==

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, ''et al''. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the ''M. mycoides'' natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

== THE SEQUENCE ==

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: GenBank. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010)

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. (Gibson, ''et al.'' 2010)

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture (~1011 cells). 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

== RESULTS ==

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

== BLUE COLONIES ==

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

== WATERMARKS ==

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson ''et al''. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

== RESTRICTION ANALYSIS ==

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. (Gibson, ''et al.'' 2010)

== SEQUENCING ==

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the ''M. capricolum''.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

== AN UNWELCOME MUTATION ==

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The dnaA mutation was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

== A PROBLEM WITH THE RESTRICTION SYSTEM ==

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

== THE SCIENTIFIC ACHIVEMENT ==

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

== THE RESPONSE ==

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

== THE ARTIFICIAL LIFE ==

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
== REFERENCES ==

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381–390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984–6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632-638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985-986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79–89.

SLEATOR, R. D., The story of ''Mycoplasma mycoides'' JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229-230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687–701.

VENTER, C. Watch me unveil "synthetic life" 18:14
http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en (available on 25. 12. 2014)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241–248

http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ (available on 25. 12. 2014)

Fact Sheet: JCVI’s Synthetic Genomics Research, http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf (available on 25. 12. 2014)

Creation of a bacterial cell controlled by a chemically synthesized genome

2014-12-28T16:19:26Z

Eva Lucija Kozak: New page: (Eva Lucija Kozak) = Introduction = == BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 == In the following paper I will present the first creation of a bacteri...

(Eva Lucija Kozak)

= Introduction =

== BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME JCVI-syn1.0 ==

In the following paper I will present the first creation of a bacterial cell controlled by a chemically synthesized genome that was done by the scientists Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang and their colleagues at the Craig Venter's laboratory in 2010 and described in the article Gibson, D. G., Glass, J. I., Lartigue, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56. They (re)created life using a digitized DNA sequence, stored in a computer file. The result was a living entity capable of growth and self replication whose parents was a computer as stated by Craig Venter (Venter, 2010). This was an important achievement for science because of the development of new technologies and as a proof that genetic information necessary for life can be stored in a digital file.
The creation of a bacterial cell controlled by a chemically synthesized genome expanded possibilities of creating artificial life and stretches the bounds of our common conception of (natural) life.
Once determining the sequence of the base pairs in a DNA molecule the information contained in the genome literally becomes digitalized. Based on this digital information a DNA molecule can be synthesized by a machine in a laboratory. Its transplantation in a host cell results in an almost synthetic cell.

== ''Mycoplasma'' ==

In this project the genome of ''Mycoplasma Mycoides'' was synthesized and transferred to ''Mycoplasma capricolum. Mycoplasma'' is genus of bacteria that lack a cell wall and have a small genome which make them easy to work with. ''Mycoplasma Mycoides'' is a parasitic micro-organism that causes major lung diseases of ruminants (cattle and goats).

== J. Craig Venter Institute ==

The research was done at the J. Craig Venter Institute which is an important and influential not-for-profit research institute in Rockville, MD and La Jolla, CA, founded by J. Craig Venter, Ph.D. It is dedicated to the advancement of the science of genomics, the understanding of its implications for society, and communication of those results to the scientific community, the public, and policy-makers.

= What made this possible? =

== BACKGROUND RESEARCH ==

There are three main methods that made this project possible: DNA sequencing, DNA synthesis and genome transplantation.
The great discovery of the DNA structure as a double helix by James Watson and Francis Crick in 1953 was followed by further analysis of DNA molecules and since 1970s it became possible to determine the sequence of base pairs in a DNA and unravel the genetic code of organisms. Further developing sequencing methods enabled more and more accurate reading of longer and longer DNA molecules. In 1977 Sanger and colleagues determined the sequence of a whole genome of a phage ϕX174. The first genetic sequence of a whole self-replicating bacterium, ''Haemophilus influenzae'' became known in 1995 and the genome of ''Mycoplasma genitalium'' was sequenced in the same year by Craig Venter's team. Since then sequencing has become much faster and less expensive and our knowledge of genomes of different organisms is increasing exponentially.

Besides determining sequences researchers also have developed methods to synthesize DNA molecules. The crucial information for synthesizing DNA is its sequence. Short DNA oligonucleotides are nowadays easily synthesized but a synthesis of longer DNA molecules still presents a challenge. It is possible to synthesize small oligonucleotides and than join them in a longer DNA molecule which was first demonstrated by Khorana and colleagues in 1970. (Gibson, et al. 2009)
The team at the Craig Venter Institute has been intensively working on the production of very long DNA molecules by assembling smaller DNA molecules. By 2008, they showed that they could produce a long DNA molecule as they synthesized an artificial chromosome of ''M. genitalium''. During their research on minimal genome project Venter's team developed a method for synthesizing DNA molecules long up to 6kb. (Gibson, ''et al.'' 2010)

For creating a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 a different task needed to be mastered as well, which was a genome transplantation. Genome transplantation is a procedure in which DNA from one species is transplanted into a cell of another species resulting in changing the recipient cell to the donor species. It is the process of installing a naked bacterial chromosome into a suitable recipient cell in such a way that the installed genome commandeers and reprograms the machinery of the recipient cell. (Glass, 2012) Researchers make this happen by fusing cells and new DNA, then allowing cells to divide and form daughter cells. At the end the cells containing the new DNA are selected and the colonies are grown. In 2007 the researchers at the Craig Venter Institute managed to successfully transplant the chromosome from one microbial species to another. For this purpose a gentle isolation of intact donor genome had to be performed and the extracted DNA from ''Mycoplasma mycoides'' was then used to replace the genome of the bacterium ''Mycoplasma capricolum'' with the native chromosome of ''Mycoplasma mycoides''. (Lartigue, ''et al.'' 2007).

Getting very close to the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 in 2009 the researchers at the Craig Venter Institute showed they could extract the M. mycoides natural chromosome, place it into yeast, modify the bacterial genome, and then transfer it to ''M. capricolum'', a close microbial relative (Lartigue, ''et al.'' 2009). This process was similar to the one used in the creation of a bacterial cell controlled by a chemically synthesized genome but in the latter the DNA molecule put in ''M. capricolum'' was produced synthetically.

== THE SEQUENCE ==

When new sequences of genomes of different organisms are determined and the information is put in a genetic bank: GenBank. GenBank is a database of all publicly available nucleotide sequences and their protein translations. This database is produced at the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

When starting the project the sequence of the ''M. mycoides'' was not completely determined yet but there were two projects working on it. Therefore the design of the synthetic ''M. mycoides'' genome was based on sequences of two laboratory strains of ''M. mycoides''. The genome of ''Mycoplasma myocides'' subspecies ''capri'' with GenBank accession code CP001621 was sequenced by Lartigue ''et al.'' This sequence of the ''M. mycoides'' strain with a length of 1 089 202 bp was the one used as the genome donor in genome transplantation mentioned earlier. In a GenBank there is another sequence of a ''M. mycoides'' GenBank accession code CP001668 – This is the sequence with a length of 1 084 586 bp of an ''M. mycoides'' strain with a deleted gene for a Type III restriction endonuclease engineered in yeast for the transplantation. (Lartigue, ''et al.'' 2009)

Consequently Gibson and his colleagues started with the draft sequences which were later corrected when the whole genome of ''Myoplasma myocides'' was determined. They started work with CP001621 but then they supplemented it with CP001668 and replaced all the previously synthesized DNA molecules that contained differences from this sequence.

= How did they synthesize it? =

The genome of ''Mycoplasma myocides'' is 1 084 586 bp long which is much more than it could be synthesized in one piece. For this project smaller oligonucleotides were synthesized and then assembled in three stages to produce bigger and bigger pieces. In vitro enzymatic methods were used to synthesize smaller parts which were then linked by in vivo homologous recombination in the yeast. The yeast ''Saccharomyces cerevisiae'' has a capacity to take up and recombine DNA fragments so it was employed to assemble the DNA in stages; the first stage involved taking 10 cassettes at a time to build 110 10 000 bp segments. In the second stage, these 10 000 bp segments were taken 10 at a time to produce 11 100 000 bp segments. In the final step, all 11 100 kb segments were assembled into a complete synthetic genome. (Sleator, 2010)

Firstly, 1080 bp long DNA molecules - cassettes were produced and verified by Blue Heron (Bothell, Washington). The cassettes had 80 bp long overhangs to adjacent cassette facilitating correctly orientated sequence assembly. Overlapping cassettes contained Not I restriction sites at their termini and could recombine in the presence of a vector. 1078 of such 1080 bp long cassettes were put in a yeast ''Saccharomyces cerevisiae'' where they recombined. Recombination is a process by which two DNA molecules in a same cell exchange genetic information and can be used to join genetic material. (Gibson, ''et al.'' 2010)

After recombination in yeast the cassettes were transferred to a bacterium E. coli. 10-kb intermediates were expected to be produced in this stage so they screened E. coli for such cassettes which was at least in 10% cases. The intermediates were isolated and sequenced for verification. The cassettes containing errors were eliminated apart from 19 polymorphic differences that appeared harmless and were not corrected. (Gibson, ''et al.'' 2010)

100-kb cassettes were designed in the next step again by recombination in yeast. Those intermediates were too big to be stable in ''E. coli'' so they were directly extracted from yeast which is a bit more complicated procedure compared to the extraction from ''E. coli''. This method produced ~1 mg of each assembly per 400 ml of yeast culture (~1011 cells). 11 assemblies produced in yeast spheroblasts, cells from which the cell wall has been almost completely removed, were isolated after alkaline-lysis. 25% or more of the screened clones were correct and one of them was chosen for further work. The extracts were treated with exonuclease to remove a few nucleotides at the end of the DNA molecules and an anion exchange column was used for purification of yeast DNA. Ion Exchange Chromatography (IEX) is a method that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. As the intermediates were still not completely clean of the yeast DNA the scientist used an interesting method. They pooled the samples of each assembly intermediates in a molten agarose. When the agarose solidified, the fibers thread through and topologically traped circular DNA, what are the intermediates for this project. The yeast DNA is linear and was therefore not trapped but removed from agarose by electrophoresis. Then the circular assembly intermediates were digested with a restriction enzyme Not I which made them linear so they could be released. Finally, the intermediates were analysed by FIGE, field inversion gel electrophoresis, which is a type of gel electrophoresis in which large molecules may move faster than the small ones. (Gibson, ''et al.'' 2010)
At the end the assemblies were multiplied by PCR and transformed into yeast spheroplasts for the final assembly of the DNA fragments into the whole genome. This stage was performed in yeast by making use of the yeast genetic systems so no additional vector was required because the yeast cloning elements were already present in one of the assemblies (811-900). Following the recombination, the colonies were screened by PCR using primer pairs designed to span each of the 11 100-kb assembly junctions and one clone (sMmYCp235) produced all amplicons. A positive control, PCR of the wild-type (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons which meant that the genome was complete. (Gibson, ''et al.'' 2010)

= How was the synthetic genome transplanted? =

The whole synthetic genome of ''Myoplasma mycoides'' was stably grown in a yeast as a centromeric plasmid. It was identical to the natural genome of ''Myoplasma mycoides'' except for the 19 polymorphic sites and the watermarks (see section WATERMARKS).
DNA was then transferred from yeast to a receptive cytoplasm of ''M. capricolum'' cell. This step of the project had been done before with a natural genome of ''Myoplasma mycoides'' being transplanted into ''Mycoplasma capricolum'' by Lartigue ''et al.'' (Lartigue ''et al.'' 2009)

The donor ''M. mycoides'' genomes were treated with calcium chloride and the ''M. capricolum'' recipient cells with polyethylene glycol (PEG). In solution, the positively charged calcium ions bind loosely to the negatively charged phosphate bonds that connect the DNA bases comprising the donor genome. Thus, the donor genome is no longer repelled by the recipient cell membranes, which are also negatively charged. The PEG changes the fluidity of the membranes, causing the cells to fuse. After transplantation a cell contains both genomes but once this heterodiploid cell is returned to growth conditions, it divides with one genome ending up in each daughter cell and the selection can be made. (Glass, 2012)

''M. mycoides'' was transformed with a vector containing a selectable tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence, for selection and propagation in yeast as a yeast centromeric plasmid (Lartigue ''et al.'' 2009).

Following the successful transplantation the synthetic genome began to encode all the proteins naturally present in ''M. Mycoides''. Among other proteins required for functioning of the cell there were also restriction enzymes which slowly degraded the native ''M. capricolum'' genome. After 30 divisions the cells did not contain any proteins that were previousely present in ''M. capricolum''. Therefore the only genome left in the cell was the one of ''M. Mycoides'' which was transplanted there. (Sleator, 2010)

== RESULTS ==

The cells containing only the synthetic genome were self-replicating and capable of logarithmic growth. The colonies on agar plates were growing in the same way as the ones of natural ''M. Mycoides'' with the colony morphology reminding of a fried egg which is characteristic of most mycoplasmas. (Gibson, ''et al.'' 2010)

The researchers did several different tests to see whether the results of the experiment truly were what they had expected in order to prove the creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0.

The morphology of the cells was compared to the one of natural ''M. Mycoides'' using an electron microscope which is a device that uses accelerated electrons as a source of illumination and can reveal structure of very small objects, like cells. The shape of the cells was examined by scanning and transmission electron micrograph.
Proteomic analysis were also made by two-dimensional gel electrophoresis to verify if the expression of proteins in the bacterial cell controlled by a chemically synthesized genome was as expected. (Gibson, ''et al.'' 2010) The only difference between the synthetic cells and the control strain was slightly faster growth of JCVIsyn1.0 detected in a color-changing unit assay. (Gibson, ''et al.'' 2010) Overall the analysis indicated that the experiment was successful.

= How did they prove it? =

== BLUE COLONIES ==

The cells were grown on a medium containing tetracycline and X-gal at 37°C. Since the ''M. mycoides'' genome was transformed with a vector containing a β-galactosidase gene the researchers could identify the successfully transformed colonies by blue colour. β-galactosidase in the cells makes a blue product out of the X-gal in the medium. The blue colonies therefore proved the cells contained the vector with synthetic genome. (Gibson, ''et al.'' 2010)

To prove that the cells indeed are controlled by a chemically synthesized genome two analyses were performed to distinguish them from natural ''M. mycoides''.

== WATERMARKS ==

The watermark is a short sequence of base pairs added to the DNA molecule to prove its synthetic origin. When synthesizing the genome Gibson ''et al.'' added 4 watermark sequences to the genome of ''M. mycoides'' on the sites that were proved or predicted not to interfere with cell viability. DNA watermark technology employs DNA sequences with encrypted information to label organisms. DNA watermark technologies are generally comprised of three processes: encryption, labelling and detection. (Yamamoto, ''et al.'' 2014) An information is encrypted in an organism by genetic engineering or when synthesized. In detection, the hidden information is mined and decrypted from the genomic sequences to obtain the original message. One of the main purposes of using the watermarks is an integration of confidential information in the DNA because the complexity of the DNA makes the decryption more difficult. Watermarks are also a reliable technology to label breeding lines. However, watermarks are mainly used as a proof of genetic modification of an organism which was also the case in this project. Gibson et al. did not have an important message to preserve nor they needed to hide secret information in the DNA. They encrypted their email addresses, names of 46 authors and other key contributors as well as three famous quotations: "To live, to err, to fall, to triumph, to recreate life out of life" from James Joyce's Ulysses; "See things not as they are, but as they might be" from American Prometheus, a biography of Robert Oppenheimer; and "What I cannot build, I cannot understand" from the writings of the physicist Richard Feynman; which they saw as suitable for their project.
Those watermarks were used to prove the synthetic nature of the genome. Primers specific to the watermarks were used to perform a PCR and the length of the PCR products matched the predicted one.

== RESTRICTION ANALYSIS ==

Another proof of the genome being synthetic was provided by restriction analysis. DNA, isolated from yeast in was restricted by two restriction enzymes: Asc I and BssH II. The restriction sites for those two enzymes were present in three of the four watermark sequences, the length of the DNA molecules after restriction was different for natural ''M. mycoides'' and the one controlled by a chemically synthesized genome which resulted in different pattern when analysed by gel electrophoresis. (Gibson, ''et al.'' 2010)

== SEQUENCING ==

The final proof was the sequencing of the genome. The results matched the intended design with the exception of eight new single-nucleotide polymorphisms which appeared during the process and a transposon insertion from ''E. coli'' (IS1, a transposon in ''E. coli''), and an 85-bp duplication (a result of a non-homologous end joining event). There were no sequences belonging to the M. capricolum.

= Troubleshooting =

As the creation of a bacterial cell controlled by a chemically synthesized genome had never been done before so the researchers had to develop completely new methods and face many obstacles.

Because of their research being orientated towards a minimal genome, at first their target organism was ''M. genitalium'', a sexually transmitted pathogen microbe of humans which has only 525 genes. However, the ''M. genitalium'' has a doubling time of 16 hours, so it was replaced by faster growing ''M. mycoides'' even though the latter has a bigger genome.

== AN UNWELCOME MUTATION ==

When the synthetic genome was initially put into ''M. capricolum'', nothing happened and it took the researcher quite a lot of time to figure what went wrong. They solved this problem by a semi-synthetic technology to clone genomes and the functionality of each 100-kb synthetic segment was tested. Parts of natural genomes and the synthetic genomes were mixed and matched to identify the part containing the mutation. Semi-synthetic genomes were transplanted and one of them, 811-900, turned out not to be viable. It contained a single–base pair deletion that created a frame-shift in dnaA, an essential gene for chromosomal replication. The dnaA mutation was than repaired and the mutated one was later used as a negative control. (Gibson, ''et al.'' 2010)

== A PROBLEM WITH THE RESTRICTION SYSTEM ==

Another problem the researchers had to face was the restriction system of ''M. capricolum''. Organisms generally have a method to protect themselves against foreign DNA. This method is a restriction system that degrades all unwelcome genetic material. Naturally a DNA molecule is protected from the restriction system by being methylated. The synthesized genome of ''M. mycoides'' was grown in yeast and was 'naked' that is unmethylated. The natural DNA sequences encoding the methylases cannot be expressed in yeast because they contain UGA tryptophan codons, which in yeast function as stop codons. Therefore some modifications were needed. A big obstacle was the fact that the donor and recipient mycoplasmas share a common restriction system which the team did not predict in advance. To solve this problem the restriction system of ''M. capricolum'' was disrupted. The single restriction enzyme in ''M. capricolum'' was inactivated by integration of a puromycin-resistance marker into the coding region of the gene. (Lartigue, ''et al''. 2009)

= Why is this important? =

== THE SCIENTIFIC ACHIVEMENT ==

The first creation of a bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 mainly presents an important proof of a concept and gives a rise to planning what more can be done. Most importantly it proved that the genetic information necessary for life can be stored in a computer file. It was recognised as “a defining moment in the history of biology and biotechnology,” by Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal Artificial Life (Pennisi, 2010). As an achievement in synthetic biology the creation of a JCVI-syn1.0 presents a potential to construct useful micro-organisms with a desired behaviour which could be used in industry, agriculture, medicine, environmential care or bioterrorism. For future scientific research this project is most important for having invented and developed new synthetic genomics techniques called genome assembly and genome transplantation. Recreation of something can be a proof of understanding it, which is often used as a motto in synthetic biology. According to Dr. Ham Smith: “With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works.”

== THE RESPONSE ==

The response to the news about the first creation of a bacterial cell controlled by a chemically synthesized genome in 2010 was huge. There were more than 500 different stories published on the internet. “It represents an important technical milestone in the new field of synthetic genomics,” said yeast biologist Jef Boeke of Johns Hopkins University School of Medicine in Baltimore, Maryland (Pennisi, 2010). Mark Bedau, a philosopher at Reed College in Portland, Oregon, and editor of the scientific journal ''Artificial Life'', labbeled the creation of the JCVI-syn1.0 “a defining moment in the history of biology and biotechnology.” (Pennisi, 2010).
The J. Craig Venter Institute is known for good communication and convincing talks for the public and their work has had a big impact on the public awareness of synthetic biology. When the public in America was asked about the recent announcement by the J. Craig Venter Institute of its creation of a partly synthetic life-form on the basis of DNA produced in a laboratory, nearly one in four (24%) adults said that they recalled hearing about it (Pauwels, 2013).
The the creation of the synthetic cell of course rised some concerns as well. Kenneth Oye, a social scientist at the Massachusetts Institute of Technology in Cambridge said: “Over the long term, the approach will be used to synthesize increasingly novel designed genomes. Right now, we are shooting in the dark as to what the long-term benefits and long-term risks will be.” (Pennisi, 2010).

== THE ARTIFICIAL LIFE ==

Anthony Forster, a molecular biologist at Vanderbilt University in Nashville, Tennessee and others emphasized that this work didn’t create a truly synthetic life form, because the genome was put into an existing cell (Pennisi, 2010). However, a bacterial cell controlled by a chemically synthesized genome essentially differs from a natural life as it's most important components were created by man. Since the creation of JCVI-syn1.0 discussions of synthetic life are no longer just conjecture. It's importance is even greater considering future research and creating of artificial life it enabled.
Technically speaking, artificial life (Alife) is an interdisciplinary field of research characterized by attempts to simulate and synthesize lifelike processes through artificial (in vitro, in silico, or in theorio) means. In 1994 Daniel Dennett urged philosophers not to consider Alife as just another phenomenon in need of critical philosophical analysis but rather as a new sort of philosophy. Dennett characterized Alife as a method rather than a phenomenon. Alife provides a wide variety of means for rethinking our conceptions of life forcing us to create new imaginative alternatives to what might be, or what could have been. (Swan, 2009)
Even though it is argued that life may be more abstract than we think, and therefore the actual manifestation of life, whether biological or theoretical, is less important (Swan, 2009), the actual creation of JCVI-syn1.0 needs a reflection. “This experiment will certainly reconfigure the ethical imagination,” said Paul Rabinow, an anthropologist at the University of California, Berkeley, who studies synthetic biology (Pennisi, 2010).

= Conclusion =

The bacterial cell controlled by a chemically synthesized genome JCVI-syn1.0 was synthesized as one of the accomplishments on the Craig Venter's path to determine a minimal genome necessary for life in a laboratory, the ideal platform for analysing the function of every essential gene in a cell. The project of creating JCVI-syn1.0 costed estimated 40 milion dollars (Pennisi, 2010) and resulted in producing a living entity capable of growth and self replication. This was an important achievement for science because of the development of new technologies and as a proof that the genetic information necessary for life can be stored in a computer file.
In this project the DNA was transplanted in an existing cell so the next step is probably the creation of a completely artificial life form. Nevertheless, JCVI-syn1.0 has been recognised as a synthetic cell and the existence of an artificial life form calls for its contextualization from a philosophical point of view as well as it is expected to extend our ideas about the possible.

= Where can I read more about this? =
== REFERENCES ==

CAMERON, D. E., ''et al.'' A brief history of synthetic biology, ''Nature Reviews Microbiology'', May 2014 Vol. 12, No 5, p. 381–390.

GIBSON, D. G., Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides, ''Nucleic Acids Research'', 2009, Vol. 37, No. 20, p. 6984–6990.

GIBSON, D. G., GLASS, J. I., LARTIGUE, C., ''et al.'' Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, ''Science'', July 2010, Vol. 329, p. 52 – 56.

GLASS, J. I. Synthetic genomics and the construction of a synthetic bacterial cell, ''Perspectives in Biology and Medicine'', Autumn 2012, Vol. 55.4, p. 473 – 89.

LARTIGUE, C., GLASS, J. I., ALPEROVICH, N. ''et al.'' Genome Transplantation in Bacteria: Changing One Species to Another, ''Science'', August 2007, Vol. 317, p. 632-638

LARTIGUE, C., VASHEE, S., ALGIRE, M. A., ''et al.'' Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast, ''Science'', 2009, Vol. 325, p. 1693 – 1696.

PENNISI, E. Synthetic Genome Brings New Life to Bacterium, ''Science'', May 2010, Vol. 328, p. 985-986.

PAUWELS, E., Public Understanding of Synthetic Biology, ''BioScience'', February 2013, Vol. 63, No. 2, p. 79–89.

SLEATOR, R. D., The story of Mycoplasma mycoides JCVI-syn1.0: The forty million dollar microbe, Bioengineered Bugs, ''Landes Bioscience'', July/August 2010, Vol. 1, No. 4, p. 229-230

SWAN, L. S., Synthesizing insight: artificial life as thought experimentation in biology, ''Biol Philos'', 2009, Vol. 24, p. 687–701.

VENTER, C. Watch me unveil "synthetic life" 18:14
http://www.ted.com/talks/craig_venter_unveils_synthetic_life?language=en (available on 25. 12. 2014)

YAMAMOTO, N., KAJIURA, H, TAKENO, S. ''et al.'', A watermarking system for labeling genomic DNA, ''Plant Biotechnology'', 2014, Vol. 31, p. 241–248

http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/ (available on 25. 12. 2014)

Fact Sheet: JCVI’s Synthetic Genomics Research, http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/fact-sheet2.pdf (available on 25. 12. 2014)

HMC

2013-11-22T19:07:25Z

Eva Lucija Kozak: New page: '''Modified handmade cloning HMC''' - modificirano ročno kloniranje, izumljeno že 2001, so P. Zhang in P. Liu idr. uporabili za produkcijo transgenskih ovc s povišanimi nivoji omega-3 m...

'''Modified handmade cloning HMC''' - modificirano ročno kloniranje, izumljeno že 2001, so P. Zhang in P. Liu idr. uporabili za produkcijo transgenskih ovc s povišanimi nivoji omega-3 maščobnih kislin. To so prve transgenske ovce narejene s HMC, je pa bila metoda pred tem že uspešno uporabljena.

Omega-3 maščobne kisline so v prehrani zelo pomembne. Idealna dieta naj bi vsebovala veliko višje razmerje omega-3 v primerjavi z omega-6 maščobnimi kislinami, kot jih vsebuje dieta v zahodnem svetu (idealno je 4:1, povprečna prehrana vsebuje 16:1).
Večina sesalcev je izgubila sposobnost sintetiziranja omega-3 maščobnih kislin, glista C. elegans pa učinkovito pretvarjanja omega-6 maščobne kisline v omega-3 maščobne kisline z desaturazo, ki je zapisana na fat-1 genu.
Fat-1 gen iz C. elegans (1209bp) so zato optimizirali za sesalce. Skonstruirali so evkariontski ekspresijski vektor z genom mfat-1, CAD promotorjem in selekcijskim markerjem za neomicin. Vektor je bil lineariziran in z elektroporacijo transficiran v ovčje fibroblaste kitajske merino ovce.
S PCR in RT-qPCR so preverjali prisotnost in nivo mfat-1 mRNA, s plinsko kromatografijo pa so ugotavljali nivoje različnih maščobnih kislin v celicah. Izbrane donorske celice so z metodo HMC združili z brezjedrnimi oocitami ter tako skonstruirali transgenske zarodke.

HMC metoda je zelo podobna že dolgo uporabljani metodi SCNT (somatic cell nuclear transfer), ki pa temelji na uporabi specializiranih instrumentov za mikromanipulacijo.

HMC POSTOPEK: Iz zaklanih živali (ovc) vzamejo jajčnike, iz katerih vzamejo oocite, kulumus celice pa odstranijo z vorteksiranjem. Oocite nato inkubirajo v demecolcinu, zonae pellucidae razgradijo z mešanico proteinaz. Enukleacijo opravijo ročno z ostrim mikrorezilom in zavržejo karioplaste (s kromatinom), citoplaste pa služijo kot receptorske celice – uporabijo 2 brezjedrni oociti za en embrij. Somatske celice druge živali (v tem primeru gensko spremenjene celice) tripsinizirajo in z elektrofuzijo združijo z receptorskimi celicami. Rekonstruirane embrije kemijsko aktivirajo in 7 dni gojijo in vitro.
Embriji so bili gojeni v modificiranem Well-of-the-Well (WOW) sistemu, ki je učinkovit pristop za izboljšanje razvoja embriov. Dozorevajo lahko blizu skupaj v stabilnem mikrookolju, kar omogočajo majhne vdolbinice na dnu petrijevke.
Po 7 dneh gojenja so zarodke na stopnji blastociste kirurško vstavili v ovce. Skotili so se 3 transgenski jagenjčki, PP-02 je poginil v 48 urah. Skupna učinkovitost: 5,7%.
Vsi transgenski jagenjčki so imeli isti genotip in v vseh so dokazali prisotnost mfat-1 gena.

Prisotnost in funkcionalnost mfat-1 gena v transgenskih jagenjčkih je bila potrjena s PCR, RT-qPCR, Southern, Northern in DNA sekvenciranjem.
Z BamHI so sekvencirali genom in s Southeren blotom dokazali eno kopijo gena, ki se je vstavila v 5. kromosomu, v intronu funkcionalnega gena za protein Cep120. Za identifikacijo mesta insercije so uporabili TAIL-PCR – Thermal asymetric interlaced PCR.
Na jagenjčku PP-02 so s plinsko kromatografijo preverili dejansko učinkovitost desaturaze – vsebnost omega-3 maščobnih kislin v različnih tkivih, ter opazili najvišje nivoje predvsem v skeletni mišici in jeziku, občutno povišani pa so bili tudi nivoji v srcu, vranici in jetrih. Vse n-3 maščobne kisline so konstruirale 6,2% vseh maščob v skeletni mišici (WT: 3,5%).
Poskus zaenkrat ni obsegal nadaljnjih raziskav dednosti in stabilnosti mfat-1 gena.

HMC-prednosti:
Metodo HMC je dokazano uporabna za več domačih živali – pujsi, govedo. v primerjavi s tradicionalnim kloniranjem HMC radikalno zniža stroške. Poveča produktivnost, omogoča zamrzovanje embriov za shranjevanje, rezultati pri številu rojstev so primerljivi ali celo boljši kot pri tradicionalnem kloniranju. Z avtomatizacijo postopka bi lahko postalo kloniranje bolj množično.
Heteroplazmičnost, ki nastane zaradi treh različnih virov mitohondrijev se v praksi še ni pokazala za problematično. Odsotnost zone pellucide kritiki izpostavljajo kot povišano možnost za prenos bolezni, vendar tudi pri SCNT celice niso manj občutljive.

ČLANEK: Zhang P, Liu P, Dou H, Chen L, Chen L, et al. (2013) Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids. PLoS ONE 8(2): e55941. doi:10.1371/journal.pone.0055941

Seminarji TehDNA

2013-11-06T21:07:03Z

Eva Lucija Kozak:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

Seznam tem za seminarje:

# Mutageneza (16.10.), 3 seminarji: 1. Urban Bezeljak (CRISPR/Cas9) 2. Uroš Stupar (ZFN nukleaze) 3. Helena Vajović (tarčna mutageneza)
# Izražanje na površini (23.10.), 3 seminarji: 1. Mitja Crček (Ribosome display), 2. Klara Tereza Novoselc (Phage display) 3. Živa Marsetič (Predstavitev na površini bakterij)
# Dvohibridni sistemi (30.10.), 3 seminarji: 1. Katja Kovačič (BiFC) 2. Barbara Žužek (YTH s knjižnico celic HeLa) 3. Bernarda Majc (YTH)
# Mutageneza/genetika (6.11.), skupaj 3 seminarji: 1. Valter Bergant (scFv phage display), 2. Ana Kapraljević (gene silencing shRNA), 3. Tjaša Blatnik (gene overexpression)
# GSO v agronomiji (12.11.), 3 seminarji: 1. Niki Bursič (izboljšanje tolerance na mraz in slanost), 2. Petra Malavašič (biofortifikacija riža), 3. Jernej Mustar (rizobakterijska simbioza)
# Transgenske živali (26.11.), 3 seminarji: 1. Andrea Grof (transgenske kokoši), 2. Eva Lucija Kozak (HMC), 3. Špela Pohleven
# Izvorne celice (3.12.), 4 seminarji: 1. Sara Primec, 2. Alja Zottel, 3. Tjaša Goričan, 4. Rok Štemberger
# DNA-diagnostika (10.12.), 4 seminarji: 1. Tina Gregorič , 2. Eva Knapič, 3. Veronika Jarc, 4. Jana Verbančič
# Forenzika, arheologija, sistematika (17.12.), 3 seminarji: 1. Matja Zalar, 2. Andreja Bratovš, 3. Maja Remškar
# Mikromreže, genomike (7.1.), 3 seminarji: 1. Andrej Vrankar, 2. Filip Kolenc 3. Nastja Štemberger
# Gensko zdravljenje s. lat. (14.1.), 3 seminarji: 1. Ana Dolinar 2. Staša Komljenovič, 3. Katarina Uršič

'''IZBIRANJE ČLANKOV ZA SEMINARJE:
Ni nujno, da je metoda, ki jo želimo predstaviti, sama tematika izbranega članka. Zaželeno je, da članek obravnava neko biološko temo, pri raziskovanju le-te pa avtorji uporabljajo metodo, ki jo želimo predstaviti.'''

'''TA TEDEN IMAMO SEMINARJE V SREDO. OD NASLEDNJEGA TEDNA NAPREJ IMAMO SEMINARJE OB TORKIH (ob 14h na dekanatu, soba D30, 1. nadstropje levo).
'''

'''POVZETKI ZA SEMINARJE 6.11.2013'''
# Valter Bergant ([[scFv phage display]])
# Ana Kapraljević ([[gene silencing shRNA]])
# Tjaša Blatnik ([[gene overexpression]])

'''POVZETKI ZA SEMINARJE 30.10.2013'''
# Katja Kovačič ([[BiFC]])
# Barbara Žužek ([[YTH s knjižnico celic HeLa]])
# Bernarda Majc ([[YTH]])

'''POVZETKI ZA SEMINARJE 23.10.2013'''
# Mitja Crček ([[Predstavitev na ribosomih]])
# Klara Tereza Novoselc ([[Phage display]])
# Živa Marsetič ([[Predstavitev na površini bakterij]])

'''POVZETKI ZA SEMINARJE 14.10.2013'''
# Urban Bezeljak ([[CRISPR/Cas9]])
# Uroš Stupar ([[ZFN nukleaze]])
# Helena Vajović ([[tarčna mutageneza]])

Seminarji TehDNA

2013-11-06T17:22:41Z

Eva Lucija Kozak:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

Seznam tem za seminarje:

# Mutageneza (16.10.), 3 seminarji: 1. Urban Bezeljak (CRISPR/Cas9) 2. Uroš Stupar (ZFN nukleaze) 3. Helena Vajović (tarčna mutageneza)
# Izražanje na površini (23.10.), 3 seminarji: 1. Mitja Crček (Ribosome display), 2. Klara Tereza Novoselc (Phage display) 3. Živa Marsetič (Predstavitev na površini bakterij)
# Dvohibridni sistemi (30.10.), 3 seminarji: 1. Katja Kovačič (BiFC) 2. Barbara Žužek (YTH s knjižnico celic HeLa) 3. Bernarda Majc (YTH)
# Mutageneza/genetika (6.11.), skupaj 3 seminarji: 1. Valter Bergant (scFv phage display), 2. Ana Kapraljević (gene silencing shRNA), 3. Tjaša Blatnik (gene overexpression)
# GSO v agronomiji (12.11.), 3 seminarji: 1. Niki Bursič (izboljšanje tolerance na mraz in slanost), 2. Petra Malavašič (biofortifikacija riža), 3. Jernej Mustar (rizobakterijska simbioza)
# Transgenske živali (26.11.), 3 seminarji: 1. Andrea Grof (transgenske kokoši), 2. Eva Lucija Kozak (somatic cell nuclear transfer SCNT), 3. Špela Pohleven
# Izvorne celice (3.12.), 4 seminarji: 1. Sara Primec, 2. Alja Zottel, 3. Tjaša Goričan, 4. Rok Štemberger
# DNA-diagnostika (10.12.), 4 seminarji: 1. Tina Gregorič , 2. Eva Knapič, 3. Veronika Jarc, 4. Jana Verbančič
# Forenzika, arheologija, sistematika (17.12.), 3 seminarji: 1. Matja Zalar, 2. Andreja Bratovš, 3. Maja Remškar
# Mikromreže, genomike (7.1.), 3 seminarji: 1. Andrej Vrankar, 2. Filip Kolenc 3. Nastja Štemberger
# Gensko zdravljenje s. lat. (14.1.), 3 seminarji: 1. Ana Dolinar 2. Staša Komljenovič, 3. Katarina Uršič

'''IZBIRANJE ČLANKOV ZA SEMINARJE:
Ni nujno, da je metoda, ki jo želimo predstaviti, sama tematika izbranega članka. Zaželeno je, da članek obravnava neko biološko temo, pri raziskovanju le-te pa avtorji uporabljajo metodo, ki jo želimo predstaviti.'''

'''TA TEDEN IMAMO SEMINARJE V SREDO. OD NASLEDNJEGA TEDNA NAPREJ IMAMO SEMINARJE OB TORKIH (ob 14h na dekanatu, soba D30, 1. nadstropje levo).
'''

'''POVZETKI ZA SEMINARJE 6.11.2013'''
# Valter Bergant ([[scFv phage display]])
# Ana Kapraljević ([[gene silencing shRNA]])
# Tjaša Blatnik ([[gene overexpression]])

'''POVZETKI ZA SEMINARJE 30.10.2013'''
# Katja Kovačič ([[BiFC]])
# Barbara Žužek ([[YTH s knjižnico celic HeLa]])
# Bernarda Majc ([[YTH]])

'''POVZETKI ZA SEMINARJE 23.10.2013'''
# Mitja Crček ([[Predstavitev na ribosomih]])
# Klara Tereza Novoselc ([[Phage display]])
# Živa Marsetič ([[Predstavitev na površini bakterij]])

'''POVZETKI ZA SEMINARJE 14.10.2013'''
# Urban Bezeljak ([[CRISPR/Cas9]])
# Uroš Stupar ([[ZFN nukleaze]])
# Helena Vajović ([[tarčna mutageneza]])