Mycoplasma Laboratorium - Synthetic Genome

Synthetic Genome

In 2010, using the methods described above, Venter and colleagues created a strain of Mycoplasma mycoides called JCVI-syn1.0 with a synthetic genome. Initially the synthetic construct did not work, so to pin point the error — which caused a delay of 3 months in the whole project — a series of semi-synthetic constructs were created. Given the fact that the natural genome worked, the cause of the failed growth was an frameshift mutation in DnaA, a replication initiation factor, which, once corrected, worked and was verified.

The construction of a cell with a synthetic genome was done to test the methodology — allowing more modified genomes to be created in the future. To minimize sources of failure, the genome was created using a natural genome as a template. Due to the large size of a genome, apart from the elements required for propagation in yeast and residues from restriction sites, several differences are present in Mycoplasma mycoides JCVI-syn1.0 notably an E.coli transposon IS1 (an infection from the 10kb stage) and an 85bp duplication.

However, the project has received heavy criticism as it claims to have created a synthetic organism. This claim arises from the fact that the genome was synthesized chemically in many pieces (a synthetic method), joined together by means of molecular biological techniques (an artificial method), and transplanted into the cytoplasm of a natural cell (after a few generations, though, the original protein content is undetectable). The two species used as donor and recipient are of the same genus as the more distant two species are, the less the correct protein interactions are maintained, such as binding factors and binding sites, which mutate together (epistasis). Consequently, Paul Keim (a molecular geneticist at Northern Arizona University in Flagstaff) notes that "there are great challenges ahead before genetic engineers can mix match, and fully design an organism's genome from scratch" due to this issue. DNA is the template for protein construction and requires protein to do so, a chicken-and-egg conundrum solved by the RNA world hypothesis, consequently synthetic naked DNA would require several protein to kick start a viable cell. In 2000 and 2002 teams synthesized replicating hepatitis C virus (about 9600 nucleotides long) and poliovirus (about 7500 nucleotides long), viruses, however, replicate by obligatorily utilising host protein expression machinery. Furthermore, whereas DNA can easily be replicated (using DNA polymerase), transcribed (using RNA polymerase), and translated (using ribosomes and many other factors) -- all in vitro, such reactions, so far, utilise cell extracts and most components have not been synthesized de novo—that is from inorganic or atomic components.