Intercellular recombination-assisted genetic evolution

Supplied with ample variation, evolution can create a wide sweep of specialization. Yet, the chance of obtaining a desirable trait in a population is directly proportional to the size of the DNA library. Accordingly, increased genetic diversity during directed evolution becomes beneficial. Given that homologous recombination allows continual reinvention of advantageous traits, aims to harness this phenomenon are inviting. Traditional uses of homologous recombination have been restrained to shorter sequences and meticulous multi-stepped in vitro procedures, a scope far insufficient for pathway or genome-wide engineering. By enhancing both the DNA transfer and homologous recombination efficiencies in conjugative bacteria, we investigated whether in-vivo DNA shuffling could be utilized in directed evolution. In order to isolate mutants with higher DNA transmission and recombination capabilities, Multiplex Automated Genomic Engineering (MAGE), was used to generate a mutant library of the pRK2 conjugative plasmid. Lambda-red recombination was implemented to target two genomic alterations. Hyperrecombinogenic RecA from P. aureginosa was knocked-in and RecD, a known inhibitor of inter-plasmid recombination, was knocked-out. Moreover, the pRK2 oriT and entry exclusion genes, trbJ and trbK, were removed to eliminate plasmid transfer competition and to amplify exchange between donors and recipients. Bacterial conjugation was used to select for enhanced recombining mutants, as well as during an induced SOS response with mitomycin C and norfloxacin. An ultra-recombinogenic strain of Escherichia coli will be isolated and characterized with a target efficiency of 10^-3. In conclusion, in-vivo conjugative DNA shuffling technology can help engineer and optimize on a more efficient and genome-wide scale.