Creation of a bacterial cell controlled by a chemically synthesized genome

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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 (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. (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 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: 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. mycoidessubspecies 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) (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 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. (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. (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 dnaA 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

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