Investigating the Effect of Manganese Mutation Rate in E.coli Strain CSH104

CREVELING 65

Under excessive amounts of hydrogen peroxide, a cell will activate genes to protect itself against oxidation or oxidative stress. Escherichia coli will react in a similar manner and activate genes that code for mntH, a manganese import protein, to protect themselves from this stress. Manganese is a redox cofactor that is thought to help protect proteins from oxidative damage. Our goal is to understand the role manganese may have in DNA damage and repair. In this study, we have knocked out the mntH gene in a reporter strain of E.coli CSH104, which is sensitive to GC to TA transversions in DNA. We utilized the Lambda Red system, a special recombination system that insert a linear piece of DNA containing the kanamycin gene into the E.coli's genomic DNA, replacing the mntH gene. E. coli strains CSH104 and DH5-α were transformed and allowed for the recombination to occur. Colonies were seen for both E. coli strains on kanamycin plates, indicating successful knockouts. Growth curves were generated for wildtype and mutants of both CSH104 and DH5-α in the presence and absence of hydrogen peroxide. The 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. Finally, we present the results of the lac reversion assay to measure mutation rates in E.coli strain CSH104. The assay works by a single base substitution in lacZ gene which changes a single amino acid, making the protein non-functional.