Racionalna zasnova minimalnih sintetičnih promotorjev za rastline - Revision history

Alminatahirovic: /* Construction of plasmids */

2021-04-19T16:01:28Z

Construction of plasmids

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	=='''Construction of plasmids'''==		=='''Construction of plasmids'''==
	Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be		Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be
	composed of any sequence). After ligation, no scar will exist between adjacent ~~assem�bly~~ fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS		composed of any sequence). After ligation, no scar will exist between adjacent fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
	terminator [3].		terminator [3].

Alminatahirovic: /* Introduction */

2021-04-19T16:01:09Z

Introduction

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	=='''Introduction'''==		=='''Introduction'''==
	Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8		Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8].

	=='''Identification of candidate transcription factor binding sites (TFBSs)'''==		=='''Identification of candidate transcription factor binding sites (TFBSs)'''==

Alminatahirovic: /* Resources */

2021-04-19T16:00:03Z

Resources

← Older revision		Revision as of 16:00, 19 April 2021
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	=='''Resources'''==		=='''Resources'''==
	# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.		# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
	# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.		# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research. 2020, 44 str. 746 - 752.
	# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.		# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
	# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.		# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.

Alminatahirovic: /* Resources */

2021-04-19T15:59:38Z

Resources

← Older revision		Revision as of 15:59, 19 April 2021
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	# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.		# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
	# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.		# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.
	# S.J. Triezenberg. K.L. LaMarco., S.L. McKnight: Functional dissection of VP16, the trans-activator of herpes simplex virus		# S.J. Triezenberg. K.L. LaMarco., S.L. McKnight: Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Deviation. 1988, 2 str. 718-729.
	immediate early gene expression. Genes Deviation. 1988, 2 str. 718-729.

Alminatahirovic: /* Resources */

2021-04-19T15:59:29Z

Resources

← Older revision		Revision as of 15:59, 19 April 2021
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	# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.		# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
	# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.		# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.
			# S.J. Triezenberg. K.L. LaMarco., S.L. McKnight: Functional dissection of VP16, the trans-activator of herpes simplex virus
			immediate early gene expression. Genes Deviation. 1988, 2 str. 718-729.

Alminatahirovic: /* Introduction */

2021-04-19T15:58:27Z

Introduction

← Older revision		Revision as of 15:58, 19 April 2021
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	=='''Introduction'''==		=='''Introduction'''==
	Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses [~~6].~~		Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8

	=='''Identification of candidate transcription factor binding sites (TFBSs)'''==		=='''Identification of candidate transcription factor binding sites (TFBSs)'''==

Alminatahirovic: /* Construction of plasmids */

2021-04-19T15:56:30Z

Construction of plasmids

← Older revision		Revision as of 15:56, 19 April 2021
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	=='''Construction of plasmids'''==		=='''Construction of plasmids'''==
	Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site)[4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS		Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be
			composed of any sequence). After ligation, no scar will exist between adjacent assem�bly fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
	terminator [3].		terminator [3].

Alminatahirovic: /* Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression */

2021-04-19T15:51:43Z

Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression

← Older revision		Revision as of 15:51, 19 April 2021
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	=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==		=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

	Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression [3].		Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

	=='''Results'''==		=='''Results'''==

Alminatahirovic: /* Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression */

2021-04-19T15:47:48Z

Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression

← Older revision		Revision as of 15:47, 19 April 2021
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	=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==		=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

	Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression [3].		Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression [3].

	=='''Results'''==		=='''Results'''==

Alminatahirovic: /* Resources */

2021-04-19T15:47:13Z

Resources

← Older revision		Revision as of 15:47, 19 April 2021
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	# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.		# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
	# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.		# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
			# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.