Organisms have evolved defense mechanisms to protect themselves against damages and mutations that may stress their DNA. There are various factors that cause these alterations, but the one particular to this research project is oxidative damage. Manganese has the ability to protect the cell’s DNA from oxidative stress and the only known import mechanism of Mn2+ is the manganese transport protein MntH. Therefore, to explore oxidative stress in this experiment, MntH was knocked out in 3 reporter strains of E. coli: CSH101, CSH102, and CSH104. To do so, the kanamycin resistant gene was inserted into the cell to replace the MntH gene. However, the presence of this antibiotic resistance gene may cause the E. coli to behave in an unpredictable manner. Thus, pCP20, which codes for a membrane -FLP (i.e., flippase), was used to excise the kanamycin resistance. The antibiotic resistance gene was successfully removed from two strains of E. coli: CSH 101, CSH102, and CSH104. The removal of MntH and the subsequent removal of the antibiotic resistance gene was monitored using PCR and gel electrophoresis. The LacZ reversion assay found that mutation rates were increased in the strains CSH101 and CSH104 when MntH was knocked out.

Effects of manganese concentration on mutation rates of E. coli

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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