Presentation Title

Investigating the DNA Protective Effect of Mn2+ Import in E. coli strain DH5-alpha and CSH 104

Faculty Mentor

Paul Lee

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 10:15 AM

Location

9-271

Session

Bio Sciences 2

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

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. We wish to understand the role manganese may have in DNA damage and repair. To study 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 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 in both of our strains of interest. A growth curve was constructed using the knockout mutant of CSH 104 and the DH5-alpha the curve showed 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 indicate that the cells may be more susceptible to rifampicin when mntH gene is not present.

Summary of research results to be presented

We are able to perform the Wanner knockout in DH5-alpha and our strain of interest CSH 104. As expected, the mntH knockout grows more slowly than wild type in the presence of hydrogen peroxide, but there is no difference in growth under normal conditions. As previously reported, mntH has a protective role against hydrogen peroxide in E. coli. 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 indicate that the cells may be more susceptible to rifampicin when mntH gene is not present.

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Nov 18th, 10:00 AM Nov 18th, 10:15 AM

Investigating the DNA Protective Effect of Mn2+ Import in E. coli strain DH5-alpha and CSH 104

9-271

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. We wish to understand the role manganese may have in DNA damage and repair. To study 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 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 in both of our strains of interest. A growth curve was constructed using the knockout mutant of CSH 104 and the DH5-alpha the curve showed 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 indicate that the cells may be more susceptible to rifampicin when mntH gene is not present.