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Academic Background

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.

replication Kenan Murphy figure page

One or more keywords matched the following items that are connected to Murphy, Kenan
Item TypeName
Concept Mycobacterium tuberculosis
Concept Mycobacterium
Concept Mycobacterium smegmatis
Academic Article Identification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis.
Academic Article Subpolar addition of new cell wall is directed by DivIVA in mycobacteria.
Academic Article Mycobacterial recombineering.
Academic Article The Oxidative Stress Network of Mycobacterium tuberculosis Reveals Coordination between Radical Detoxification Systems.
Academic Article N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis.
Academic Article Structural and Genetic Analyses of the Mycobacterium tuberculosis Protein Kinase B Sensor Domain Identify a Potential Ligand-binding Site.
Academic Article ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes.
Academic Article Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.
Academic Article The structure of the endogenous ESX-3 secretion system.
Academic Article A natural polymorphism of Mycobacterium tuberculosis in the esxH gene disrupts immunodomination by the TB10.4-specific CD8 T cell response.
Academic Article Oligo-Mediated Recombineering and its Use for Making SNPs, Knockouts, Insertions, and Fusions in Mycobacterium tuberculosis.
Academic Article Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice.
Academic Article Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy.
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  • Mycobacterium