Construction of a Bacterial Cell that Contains Only the Set of Essential Genes Necessary to Impart Life.

摘要

We have made significant progress on all fronts of the project. On the Top Down approach to genome minimization, we have used the Essential (E), Non-essential (N), and Impaired (I) gene categories to make steady progress with gene and gene cluster deletions. To date, we have removed approximately 283 kb from the Mycoplasma mycoides JCVI-syn1.0 genome by this method. The resultant 795 kb genome is viable and grows with a normal growth rate. Our main effort has been focused on the Bottom Up approach. Results are substantial. We have continued work on the reduced genome design (RGD) based on the N/E/I categorization system and completed testing on all 8 segments from the RGD design. All segments are viable in a 7/8th wild type background. In several experiments, we have also tested genomes containing various combinations of minimized segments. We have multiple genomes with four or more minimized segments that are viable. The smallest genome to date is RGD 24678, at 696 kb. The effort to modularize the genome is in progress. A tRNA module was constructed and inserted into the M. mycoides syn1.0 genome. The resulting genome is viable following transplantation. Construction of segments with the natural tRNA loci deleted is in progress. Preliminary work aimed at genome complementation has continued. The use of a knock-in strain for re-inserting PCR amplified genes is under discussion as an approach.