Presentation Title

Analyzing the mutation rate in E. coli’s reporter strain CSH101

Faculty Mentor

Dr. Paul Lee

Start Date

17-11-2018 9:30 AM

End Date

17-11-2018 9:45 AM

Location

C162

Session

Oral 2

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Hydrogen peroxide causes DNA damage resulting in the activation of the OxyR gene which activates mntH, leading to an uptake of manganese (Mn2+). MntH codes for the the Mn2+ import pump. Knocking out the mntH gene stops the influx of Mn2+ resulting in the inability for the cell to protect itself from hydrogen peroxide. The Lambda-Red recombination method was used to knock out the mntH gene in the CSH101 strain of Escherichia coli (E.coli). CSH101 detects transversion mutation of A:T to C:G using the lac reversion assay. The lac assay allows the study of mutagenesis by observing colonies on lactose plates and LB plates. A single base substitution in the lacZ gene causes a change in amino acid making the gene nonfunctional. More colonies present on lactose plates compared to WT should show that there is a reverse point mutation within the bacteria. The gel electrophoresis of the knockout of mntH showed that there was no band that corresponds to the wild type gene (mntH gene), therefore the knockout of the mntH was successful in CSH101. Our goal is to grow CSH101 on lactose to observe the mutation rates.

Summary of research results to be presented

We are able to perform the Wanner Knockout in CSH104 and CSH101. In both strains, there is no difference in growth under normal conditions, but when the DH5-a strain and the CHS104 was tested under H2O2 conditions, there were delayed growth. Our goal for this research is to generate and characterize a knockout strains of mntH in E.coli. We were successful in creating the insert that would replace the mntH gene and transforming CSH104 and CSH101 with pkD46 which contained the genes that will carry out the recombination. We then transformed CSH104 and CSH101 and allowed for the recombination to occur. Colonies were seen for both E.coli strains on Kanamycin plates, indicating successful knockouts. The knockout was verified using PCR and we were able to prove this because we saw a band that corresponded to the kanamycin gene but not the mntH gene. Growth curves were generated for wildtype and mutants of CSH104 and in the presence and absence of hydrogen peroxide. The results indicate the mntH knockout grows more slowly than the wildtype in the presence of hydrogen peroxide. The colonies on the LB and lac plates indicated that there were mutations; meaning there was a mutation within the bacteria allowing it to consume lactose. Mn2+ may be protecting the cell from oxidative stress induced mutations.

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Nov 17th, 9:30 AM Nov 17th, 9:45 AM

Analyzing the mutation rate in E. coli’s reporter strain CSH101

C162

Hydrogen peroxide causes DNA damage resulting in the activation of the OxyR gene which activates mntH, leading to an uptake of manganese (Mn2+). MntH codes for the the Mn2+ import pump. Knocking out the mntH gene stops the influx of Mn2+ resulting in the inability for the cell to protect itself from hydrogen peroxide. The Lambda-Red recombination method was used to knock out the mntH gene in the CSH101 strain of Escherichia coli (E.coli). CSH101 detects transversion mutation of A:T to C:G using the lac reversion assay. The lac assay allows the study of mutagenesis by observing colonies on lactose plates and LB plates. A single base substitution in the lacZ gene causes a change in amino acid making the gene nonfunctional. More colonies present on lactose plates compared to WT should show that there is a reverse point mutation within the bacteria. The gel electrophoresis of the knockout of mntH showed that there was no band that corresponds to the wild type gene (mntH gene), therefore the knockout of the mntH was successful in CSH101. Our goal is to grow CSH101 on lactose to observe the mutation rates.