Biological responses to stress manifest in many ways, both as a function of the diversity with which the environment can challenge cells and organisms, as well as the multiple genetic solutions encoding tolerance mechanisms. We applied a simple osmotic stress challenge to a population of engineered E. coli cells to rapidly validate known and discover new loci implicated in salt stress tolerance.
The null mutant has long been a reliable phenotype in genomics, but why limit yourself when you can do so much more? We used Onyx technology to comprehensively survey the entire E. coli genome with a combination of novel knock- out and promoter libraries for a total of seven unique edits on all 4,200 loci.
Onyx libraries targeting 4,200 genome-wide loci for knockout or promoter substitution
With approximately 25,000 specific variants in hand, we probed the population with a titrated salt challenge and tracked plasmid barcodes for every library member across 300,000 data points. By mapping clear enrichment and depletion patterns back to gene functional annotations and biological processes, we were able to rapidly validate our experiment with the performance of known salt stress tolerance loci, and more importantly, to discover new gene functions by assigning comparable patterns to numerous additional genes.
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