Under excessive amounts of hydrogen peroxide a cell will activate genes to protect 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 by taking the place of iron in iron-containing enzymes, thus protecting them from further oxidation. It is likely that this protective effect extends to DNA repair proteins which also contain iron. Henceforth, we wish to understand the role manganese may have in DNA damage and repair. To study this, we have attempted to knock out the mntH gene in a reporter strain of E. coli which is sensitive to DNA mutation. To do this, we are using the Lambda Red system, an exogenous recombination system, to try to replace the mntH gene located on the E. coli genome with a linear DNA insert containing the kanamycin gene. For this reporter strain, growth on lactose (lac) media plates is an indicator of high mutation rates. Using the new strain of E. coli without the mntH, we will observe how many colonies occur on lac media plates and compare this number to the reporter strain of E. coli that does not have the gene deleted. These experiments will allow us to have a greater understanding of the role manganese has in E. coli. Thus far we have been successful in creating the insert that would replace the gene and transforming the reporter strain with a plasmid containing the recombination system, as evidenced by growth on the correct antibiotic selection plates, and by checking for the plasmid using PCR. We are currently in the process of transforming the bacteria with the insert that will replace mntH and allowing the recombination to occur.

Studying the effect of manganese import on mutation rates in Escherichia coli

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.