Under excessive amounts of hydrogen peroxide, a cell will stimulate genes to defend itself against oxidation. In Escherichia coli, one of these genes codes for mntH, a manganese import protein. Manganese is a redox cofactor and is thought to help protect proteins from oxidative damage. We hope to grasp the role manganese may have in DNA damage and repair. To investigate this, we have knocked out the mntH gene in the reporter strain CSH 104 and the commercially available strain DH5-alpha, which is competent to DNA transformation. We successfully used the Lambda-Red system, a unique recombination system, to insert a linear piece of DNA containing the kanamycin gene into the E.coli's genomic DNA, replacing the mntH gene in both of our strains of interest. A growth curve was designed using the knockout mutant of CSH 104 and the DH5-alpha. The curve demonstrated that the knockout grew much slower than the wild types due the lack of mntH gene. We used a rifampicin reversion assay on this strain to examine the mutation rate with two different plating volumes, 50 and 500 uL of cells. The results suggest that the cells may be more susceptible to rifampicin when the mntH gene is not present.

Investigating the DNA protective effect of Mn2+ import in E.coli strain DH5-alpha and CSH 104

255th National Meeting (2018)

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