Creating and Characterizing an mntH knockout mutant in E. coli

BSC-Ursa Minor 64

Under excessive amounts of hydrogen peroxide a cell will activate genes to protect itself against oxidation. In Escherichia coli, one of these genes, mntH, codes for a manganese import protein. Manganese is a redox cofactor and is thought to help protect proteins from oxidative damage. We wish to comprehend the role manganese may have in DNA damage and repair. To study this, we have successfully knocked out the mntH gene in a reporter strain of E. coli which is susceptible to DNA mutation. We used the Lambda Red system, a special recombination system, to insert a linear piece of DNA containing the kanamycin gene into the E. coli's genomic DNA, replacing the mntH gene. To optimize our technique, we successfully performed the knockout in a commercially available E. coli strain DH5-α, known to efficiently accept foreign DNA. We managed to generate a growth curve of DH5-alpha of the wild type and knockout mutant. Our results indicate that the mntH knockout grows more slowly than the wild type in the presence of hydrogen peroxide, which supports our hypothesis that mntH protects E. coli against oxidative stress.