Question

The successful construction and transplantation into a cell of a synthetic Mycoplasma genome was an important milestone in synthetic biology. Briefly describe the approach used in this project.

Answer 1

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.

Bacterial genome transplantation

Transfer 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

Synthesis → 1kbp: The genome sequence was synthesized by Blue Heron in 1,078 1080bp cassettes with 80bp overlap and NotI restriction sites (inefficient but infrequent cutter).

Ligation → 10kbp: 109 groups of a series of 10 consecutive cassettes were ligated and cloned in E. coli on a plasmid and the correct permutation checked by sequencing.

Multiplex PCR → 100kbp: 11 Groups of a series of 10 consecutive 10kbp assemblies (grown in yeast) were joined by multiplex PCR, using a primer pair for each 10kbp assembly.

Isolation and recombination → secondary assemblies were isolated, joined and transformed into yeast spheroplasts without a vector sequence (present in assembly 811-900)

Structure of synthetic genome

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