Mycoplasma laboratorium

Mycoplasma laboratorium or Synthia refers to a plan to produce a synthetic strain of bacterium. The project to build the new bacterium has evolved since its inception. Initially the goal was to identify a minimal set of genes that are required to sustain life from the genome of Mycoplasma genitalium, and rebuild these genes synthetically to create a "new" organism. Mycoplasma genitalium was originally chosen as the basis for this project because at the time it had the smallest number of genes of all organisms analyzed. Later, the focus switched to Mycoplasma mycoides and took a more trial-and-error approach. To identify the minimal genes required for life, each of the 482 genes of M. genitalium was individually deleted and the viability of the resulting mutants was tested. This resulted in the identification of a minimal set of 382 genes that theoretically should represent a minimal genome. In 2008 the full set of M. genitalium genes was constructed in the laboratory with watermarks added to identify the genes as synthetic. However M. genitalium grows extremely slowly and M. mycoides was chosen as the new focus to accelerate experiments aimed at determining the set of genes actually needed for growth. In 2010, the complete genome of M. mycoides subsp. capri GM12 was successfully synthesized from a computer record and transplanted into an existing cell of Mycoplasma capricolum that had its DNA removed. It is estimated that the synthetic genome used for this project cost US$40 million and 200 man-years to produce. The new bacterium was able to grow and was named JCVI-syn1.0, or Synthia. After additional experimentation to identify a smaller set of genes that could produce a functional organism, JCVI-syn3.0 was produced, containing 473 genes. 149 of these genes are of unknown function. Since the genome of JCVI-syn3.0 is novel, it is considered the first truly synthetic organism.

Minimal genome project

The production of Synthia is an effort in synthetic biology at the J. Craig Venter Institute by a team of approximately 20 scientists headed by Nobel laureate Hamilton Smith and including DNA researcher Craig Venter and microbiologist Clyde A. Hutchison III. The overall goal is to reduce a living organism to its essentials and thus understand what is required to build a new organism from scratch. The initial focus was the bacterium M. genitalium, an obligate intracellular parasite whose genome consists of 482 genes comprising 582,970 base pairs, arranged on one circular chromosome (at the time the project began, this was the smallest genome of any known natural organism that can be grown in free culture). They used transposon mutagenesis to identify genes that were not essential for the growth of the organism, resulting in a minimal set of 382 genes. This effort was known as the Minimal Genome Project.

Choice of organism

Mycoplasma

Mycoplasma is a genus of bacteria of the class Mollicutes in the division Mycoplasmatota (formerly Tenericutes), characterised by the lack of a cell wall (making it Gram negative) due to its parasitic or commensal lifestyle. In molecular biology, the genus has received much attention, both for being a notoriously difficult-to-eradicate contaminant in mammalian cell cultures (it is immune to beta-lactams and other antibiotics), and for its potential uses as a model organism due to its small genome size. In 1996, after comparing M. genitalium with another small bacterium Haemophilus influenzae, Arcady Mushegian and Eugene Koonin had proposed that there might be a common set of 256 genes which could be a minimal set of genes needed for viability. The choice of genus for the Synthia project dates to 2000, when Karl Reich coined the phrase Mycoplasma laboratorium.

Other organisms with small genomes As of 2005, Pelagibacter ubique (an α-proteobacterium of the order Rickettsiales) has the smallest known genome (1,308,759 base pairs) of any free living organism and is one of the smallest self-replicating cells known. It is possibly the most numerous bacterium in the world (perhaps 1028 individual cells) and, along with other members of the SAR11 clade, are estimated to make up between a quarter and a half of all bacterial or archaeal cells in the ocean. It was identified in 2002 by rRNA sequences and was fully sequenced in 2005. It is extremely hard to cultivate a species which does not reach a high growth density in lab culture. Several newly discovered species have fewer genes than M. genitalium, but are not free-living: many essential genes that are missing in Hodgkinia cicadicola, Sulcia muelleri, Baumannia cicadellinicola (symbionts of cicadas) and Carsonella ruddi (symbiote of hackberry petiole gall psyllid, Pachypsylla venusta) may be encoded in the host nucleus. The organism with the smallest known set of genes as of 2013 is Nasuia deltocephalinicola, an obligate symbiont. It has only 137 genes and a genome size of 112 kb.

Techniques Several laboratory techniques had to be developed or adapted for the project, since it required synthesis and manipulation of very large pieces of DNA.

Bacterial genome transplantation In 2007, Venter's team reported that they had managed to transfer the chromosome of the species Mycoplasma mycoides to Mycoplasma capricolum by:

isolating the genome of M. mycoides: gentle lysis of cells trapped in agar—molten agar mixed with cells and left to form a gel—followed by pulse field gel electrophoresis and the band of the correct size (circular 1.25Mbp) being isolated; making the recipient cells of M. capricolum competent: growth in rich media followed starvation in poor media where the nucleotide starvation results in inhibition of DNA replication and change of morphology; and polyethylene glycol-mediated transformation of the circular chromosome to the DNA-free cells followed by selection. The term transformation is used to refer to insertion of a vector into a bacterial cell (by electroporation or heatshock). Here, transplantation is used akin to nuclear transplantation.

Bacterial chromosome synthesis In 2008 Venter's group described the production of a synthetic genome, a copy of M. genitalium G37 sequence L43967, by means of a hierarchical strategy:

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