An E coli whole Genome Mutant Library Enables Rapid Selection for Improved Fitness Under Diverse Stresses

In this work we developed an E coli mutant library with saturated mutagenesis on functional sites of proteins involved in Transcription, Translation and Regulation across the E coli genome. Successful use of E coli for synthetic biology and metabolic engineering applications is impeded by stresses due to altered growth environment and/or internal metabolic burden. Data analysis from Adaptive Laboratory Evolution studies helped us understand importance of proteins controlling protein expression towards enabling tolerance for diverse stresses. We use CRISPR enabled Trackable Genome Engineering (CREATE) to build our mutant library. We also optimized various components associated with the CREATE system to generate cleaner mutant library. Targeting protein expression enables complex rewiring in protein expression. We demonstrate the ability of this approach to identify multiple mutations that enable tolerance to a particular stress. We also demonstrate the versatility of the application of our library by highlighting results from multiple selection experiments.