Presentation Title

Removing the antibiotic resistance gene from Wanner-generated MntH knockout.

Faculty Mentor

Paul Lee

Start Date

23-11-2019 8:45 AM

End Date

23-11-2019 9:30 AM

Location

62

Session

poster 2

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Organisms have evolved defense mechanisms to protect themselves against damages and mutations that may cause oxidative stress to 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 MntH. Therefore, to explore these properties of oxidative stress in this experiment, the manganese-transport protein was knocked out in strains of E. coli, CSH102 and CSH103. To do so, the kanamycin resistant gene was inserted from 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, using PCP20, which codes for a membrane -FLP (ie flippase), was used to excise the kanamycin resistance gene via site-specific recombination. The antibiotic resistance gene was successfully removed from two strains of E. coli: CSH102 and CSH103. The removal of mntH and the subsequent removal of the antibiotic resistance gene was monitored using PCR and gel electrophoresis. Further explorations regarding this topic include sequencing all CSH strains in order to confirm the clean knockout of the mntH gene.

This document is currently not available here.

Share

COinS
 
Nov 23rd, 8:45 AM Nov 23rd, 9:30 AM

Removing the antibiotic resistance gene from Wanner-generated MntH knockout.

62

Organisms have evolved defense mechanisms to protect themselves against damages and mutations that may cause oxidative stress to 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 MntH. Therefore, to explore these properties of oxidative stress in this experiment, the manganese-transport protein was knocked out in strains of E. coli, CSH102 and CSH103. To do so, the kanamycin resistant gene was inserted from 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, using PCP20, which codes for a membrane -FLP (ie flippase), was used to excise the kanamycin resistance gene via site-specific recombination. The antibiotic resistance gene was successfully removed from two strains of E. coli: CSH102 and CSH103. The removal of mntH and the subsequent removal of the antibiotic resistance gene was monitored using PCR and gel electrophoresis. Further explorations regarding this topic include sequencing all CSH strains in order to confirm the clean knockout of the mntH gene.