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

摘要

We now have a viable reduced genome design (RGD) cell that is approximately 597 kb. This is a genome in which seven of the eight genome segments are minimized. It grows at less than half the rate of wild type Mycoplasma mycoides. This genome is comprised of minimized segments 1 and 4 (from the RGD2 design), minimized segments 2, 3*, 6, 7, and 8 (from the RGD1 design), and a wild-type, non-minimized segment 5. The 3* designation denotes that this segment was derived from the RGD1 design, with three genes involved in glycerol metabolism added back into the sequence. The key issue that we must resolve to produce a fully minimized RGD is that in RGD2 segment 5 there are one or more instances of what we call synthetic lethal deletions that we have to identify and add back to the genome. A synthetic lethal is the result of deleting both members of a pair of genes that both encode the same essential function. Individually, either member of the pair can be deleted; however a genome with both genes omitted will not support life. We are currently working to identify the remaining synthetic lethals so that RGD2 segment 5 can be redesigned and synthesized. The top down approach of iteratively removing non- essential genes from M. mycoides syn1.0 continues. Genes or sets of genes are deleted from previous top down constructs while the genomes are parked in yeast cells. Then the reduced genomes are isolated from yeast cells and transplanted to determine if the deletion is viable or has an altered phenotype. This effort is more about directing the bottom up approach by identifying potential synthetic lethals than about eventually reaching a fully minimized genome by iterative gene deletion. Currently we are working on our 20th iteration of this process. The smallest genome the top down effort has made to date has 743 kb and is missing 240 protein coding genes. This deletion mutant grows normally.