(188au) Deep Scanning Mutagenesis on the Escherichia coli Genome Help Understand Principles of Protein Engineering Towards Strain Optimization

CRISPR-cas9 mediated genome engineering now provide capabilities for exploration of mutational landscapes of proteins on the genome towards strain engineering applications in E coli. However, underlying rules for approaching protein modifications for developing complex resistance traits are not clearly known. Using a resistome database for Escherichia coli, we identified mutational hot-spots on the genome. Interestingly, the hotspots are in naturally conserved essential proteins involved in regulation, transcription and cell-shape. We developed the CRispr-mediated genomic Error Prone Editing technology, that provides a platform for high resolution error-prone mutagenesis on the genome. We aim to perform deep scanning mutagenesis of the mutational hot-spots identified from the resistome. We then use next-gen sequencing towards mutation to fitness mapping of several mutations in these proteins in several stress conditions. With the mapping, we aim to identify the diversity in adaptive paths towards fitness in similar stresses. Secondly, we aim to understand the role of experimental design in selection for the mutations and associated adaptive paths. Finally, we aim to measure the stability of the chosen adaptive paths in several different environments. We want to explore the flexibility of the mutational space in the identified hot-spots, which can help us devise some rules towards complex phenotype engineering in synthetic biology and metabolic engineering applications.