Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome Gibson et al. (2010)

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1 Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome Gibson et al. (2010) In 1977 Sanger and his colleagues were the first who sequenced the complete DNA genome of a phage. From 1977 on the ability to read the genetic sequence of a wide range of species has increased exponentially. In 1995 Gibson and his colleagues read the first complete genetic sequence of a self-replicating bacterium. In 2010 already more than 1000 genomes of different species were sequenced. Nowadays it takes only a few days to sequence a whole genome, for example it takes 1-2 days to sequence a complete human genome. This was only possible by the combination of new studies in next generation sequencing and the development in cheaper and more accurate methods for synthesis of longer DNA-Molecules in synthetic biology. So we are able to read the biological information which are defining life and we are able to safe them as digital data. But couldn t it be also the other way round? Can we reproduce a complete genetic system by chemical synthesis if we just have the digitized DNA sequence? This was the question which was driving the research group of Gibson for this project. The idea was to assemble small DNA cassettes which were assembled to larger DNA molecules later on and finally to a complete genome. The whole process was subdivided into 4 steps. The synthesis of small oligonucleotides, their assembly to 1kb, 10kb and finally to 100kb assemblies, the cloning and the transplantation into a recipient cell which is controlled by the synthetic DNA. There were several hurdles during this project which they needed to overcome. At first they had to develop and to optimize new techniques for extracting an intact genome. Also they wanted to transplant a whole genome from a donor to a recipient cell and to clone entire chromosomes. They decided to use Mycoplasma mycoides subspecies as a donor cell and M. capricolum as a recipient cell. At first a sequence was designed. They designed their sequence based on a sequence of a subspecies of M. mycoides capri. This sequence was recently highly accurate finished, so there was a high dependency on the exactness of this sequence. They also designed 4 watermark sequences. Watermarks are sequences which encode unique identifiers without affecting the viability. This helps to differentiate between the synthetic genome and the natural one. The whole genome got a size of 1.08 mega-base pair and because this is too large for synthesis, the genome was built in 4 steps of 1kb, 10kb, 100kb and the complete assembly. So they started with the design of 1080bp cassettes. These cassettes were individually synthesized and sequence-verified by

2 Blue Heron, a gene synthesis company from Bothell in Washington. Of course they don t give any information about how they are synthesizing the DNA sequences. The cassettes contain Not I restriction sites for an easier screening and are recombined by vectors. They established a strategy to assemble the whole genome in 3 stages. In every step the intermediates become assembled by transformation and homologous recombination in Saccharomyces cerevisiae. Figure 1 synthetic genome based on sequence of M. mycoides To give you an overview over this assembly strategy and the size the synthetic genome of M. mycoides is shown in figure 1. The oligonucleotides are synthesized and the 1kb-intermediates are assembled(the orange arrows). The next steps are the assembly of the 10 kb intermediates in yeast (the blue arrows) and the 100kb. In the end there will be the complete genome with the 4 watermarks on it to identify the synthetic one. The cassettes and the vector were recombined in yeast to build the 10kb intermediates. Then they were transferred to E.coli with electroporation. Then they are cultivated and individual clones are selected to screen for the correct insert. They were digested with Not I and successfully seperated by gel electrophoresis.

3 The next step is the assembly of the 100kb intermediates. Therefore the 10kb assemblies were purified and pooled. The pooled cassettes were then transformed into yeast together with their respective cloning vector. Then the 100kb intermediates are produced by homologous recombination. To check the presence of all intermediates, a multiplex PCR was design with primers for every intermediate. Multiplex PCR enables a rapid detection of different targets in a sample and a single PCR experiment. Furthermore a gel electrophoresis was performed to check the size of the intermediates. For the final step of the assembly, µg-quantities of the 100kb intermediates were isolated by alkaline-lysis. For further purification they were treated with exonucleases and passed through an anion-exchange column. For further analysis a small amount of the total plasmid was digested with Not I. The digested assemblies were analyzed by field-inversion gel electrophoresis. Field-inversion gel electrophoresis can separate fragments up to megabase-pairs. It works with a uniform electric field which is periodically inverted. The puls in the forward direction got the higher field or the longer duration or both then the one in the reverse direction. Thereby it leads to net migration. To remove all of the linear yeast DNA another method is used. For the further enrichment of the circular assembly intermediates, the samples of each assembly were pooled and mixed with molten agarose. When the agarose becomes solid, agarose fibers occur which trap the circular DNA. The linear DNA is untrapped and can be separated out of the agarose by electrophoresis. To release the trapped DNA the circular intermediates were digested with Not I. They were extracted from the agarose and transformed into yeast spheroblasts. They used multiplex PCR to screen if the genome was complete. Therefore they used 11 primer pairs which are specific for each of the 11 10kb Intermediates. To show that the genome is only built of synthetic DNA, the complete genome was digested by Asc I and BssH II. Because these have the restriction sites of the watermarks which are only on the synthetic DNA. DNA which was isolated from the agarose plug was used for transplantation. The synthetic DNA was transplanted into restriction-minus M. Capricolum cells. Restriction minus means with a knockout of the restriction mechanisms. Because the cells can only stable replicate one genome, one of two genomes, the synthetic or the original one, will get lost during the replication process. The cells with the synthetic genome are selected by the X-Gal system. The cells are cultivated on a tetracycline and X-Gal medium. The synthetic DNA contains the lac Z gene which encodes for the ß-galactosidase. The ß-galactosidase can convert X-Gal into a blue compound. To be completely sure, the blue colonies were isolated and analyzed with multiplex PCR, if the DNA got the specific watermarks. For further analysis the DNA also got digested with ASC I and BssH II

4 To sum up a complete replacement of the M. capricolum genome by a synthetic genome could be confirmed. To test if each of the 100kb intermediates were functional, semisynthetic genome assembly was done. Therefore semisynthetic genomes were produced which contain a mix of natural and synthetic DNA, so the functionality could be varified without sequencing. Furthermore could be shown that none of each synthetic fraction contains any lethal mutation. The produced cells were shown to be self-replicating and capable of logarithmic growth. They also showed almost identical pattern of the protein spots. When the colonies were plated out their rates and the morphology was that of M. mycoides, not of M. capricolum. So it could be shown that based on a synthetic genome there could be generated a new living cell. Furthermore it shows that the digitalized sequences were quiet accurate, almost enough to define the important properties of the cell. So this is the proof of producing cells based on computer-designed genome sequences. It shows us that the DNA sequencing of cells and their genome allows us to safe and store life as a digital data. But where does it lead us when the synthesis costs will decrease combined with increasing automation?

5 Sources Field inversion gel electrophoresis with different pulse time ramps.; C. Heller and F. Pohl (1990) Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast.; Lartiguec et al. (2009) SOM for Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome; Gibson et al. (2010) Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome; Gibson et al. (2010)

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