Ph. D. (1983) University of Maryland
Recombineering technology for gene replacement in bacterial pathogens
Identification of Drug Targets in M. tuberculosis - creating regulatable strains for use in whole cell screens with small molecules
My work invovles the use of Red recombineering technology for gene replacement in bacterial pathogens. My lab was the first to show that the lambda Red recombination system promotes gene replacement of electroporated linear DNA substrates into the Escherichia coliK-12 chromosome at a very high efficiency (Murphy, 1998). The system is also useful in pathogenic species of E. coli (Murphy & Campellone, 2003). Work continues to improve recombinering technology in pathogens such as Pseudomonas aeruginosa and Mycobacterium tuberculosis by expression of Red-lke recombination systems from phage known to infect these hosts.
My lab is also interested in the mechnaism of the bacteriophage lambda Red recombination system. The system consist of two proteins, the ssDNA annealing Bet protein and the 5’-3’ dsDNA lambda exonuclease. These two proteins form a complex in vitro, and are thought to interact with each other in vivo. We have isolated various mutants of Bet that are deficient for both recombination and recombineering, and some that are deficient for one but not the other.
Proposed mechanism of Red Recombineering
Lambda Exo's 5' exonuclease activity (red trapezoid)) generates ssDNA, which serves as a substrate for lambda Bet (blue oligomeric ring) to bind and promote annealing to ssDNA in the lagging strand of a replication fork. This structure is stabilized by the lambda Beta protein, unitl another fork comes by and generates both a wild type and a recombinant chromosome.