CRISPR-based genome engineering to obtain combinatorial mutants in hydrogenase enzymes

While chemiosmotic theory is foundational, alternative view have been developed suggesting local energy combinations within membranes or dual-ion models, validated in fermentative conditions. Hydrogenases and respiratory complex I with discovered homology play a crucial role in bacterial bioenergetics, ion exchange homeostasis, antibiotic tolerance and thus having important role in biotechnological applications. The primary objectives of the research stay involve using CRISPRbased genome editing techniques to construct E. coli mutants with defects in hydrogenases and further investigation of mutations effects on cellular bioenergetics and pmf generation. The proposed methodology employs the CRISPR-FRT (flippase recognition target) system developed in the host organization for targeted gene knockout within the E. coli Keio collection. This approach, coupled with advanced tools such as HPLC, GC, and GFP-based sensors, allows for a comprehensive examination of mutant effects on metabolite generation, antibiotic tolerance, intracellular pH, and ATP:ADP ratio. The main outcome is that CRISPR-FRT protocol and ratiometric sensor approach for in vivo, single-cell physiology will be taken back to Armenia.