Event Title

Monitoring the Overexpression of Stress Responsive Transcription Factors in E. coli via Real-TimePCR

Presenter Information

Emily Curley, Oberlin College

Location

Science Center, Bent Corridor

Start Date

10-2-2015 12:00 PM

End Date

10-2-2015 1:20 PM

Poster Number

12

Abstract

In E. coli, there are several different signaling pathways that are activated in response to external stress triggers. These signaling pathways are governed by transcription factors, which initiate the transcription of certain subsets of genes that are responsible for countering the effects of misfolded or aggregated proteins or contributing to the synthesis and regulation of the flagella machinery. The stress-responsive transcription factor RpoE is activated in response to stressors in the bacterial periplasm, and induces the transcription of chaperones, folding enzymes, and/or degradation machinery to help restore homeostasis in the cell. We aim to clone and overexpress the RpoE transcription factor in E. coli to monitor the effects of overexpression on bacterial biofilms. Biofilms are a stage in the lifecycle of bacteria that pose problems in hospital and other settings because of their intense resistance to existing antibiotics. Overexpressing the ideal transcription factor to an optimal extent could lead to enhancing cells’ susceptibility to antibiotics by reducing the amount of biofilm formed, thereby enhancing the ability of current antibiotics to penetrate these bacterial biofilms. A plasmid containing the RpoF transcription factor under the control of the inducible arabinose promoter has been successfully cloned into E. coli cells. We are currently overexpressing RpoF and monitoring the effects of the downstream gene targets of this transcription factor on biofilm formation. To quantify the degree of up regulation of the downstream gene targets at the mRNA transcript level quantitative PCR (qPCR) is used.

Major

Neuroscience

Project Mentor(s)

Lisa Ryno, Chemistry and Biochemistry

This document is currently not available here.

Share

COinS
 
Oct 2nd, 12:00 PM Oct 2nd, 1:20 PM

Monitoring the Overexpression of Stress Responsive Transcription Factors in E. coli via Real-TimePCR

Science Center, Bent Corridor

In E. coli, there are several different signaling pathways that are activated in response to external stress triggers. These signaling pathways are governed by transcription factors, which initiate the transcription of certain subsets of genes that are responsible for countering the effects of misfolded or aggregated proteins or contributing to the synthesis and regulation of the flagella machinery. The stress-responsive transcription factor RpoE is activated in response to stressors in the bacterial periplasm, and induces the transcription of chaperones, folding enzymes, and/or degradation machinery to help restore homeostasis in the cell. We aim to clone and overexpress the RpoE transcription factor in E. coli to monitor the effects of overexpression on bacterial biofilms. Biofilms are a stage in the lifecycle of bacteria that pose problems in hospital and other settings because of their intense resistance to existing antibiotics. Overexpressing the ideal transcription factor to an optimal extent could lead to enhancing cells’ susceptibility to antibiotics by reducing the amount of biofilm formed, thereby enhancing the ability of current antibiotics to penetrate these bacterial biofilms. A plasmid containing the RpoF transcription factor under the control of the inducible arabinose promoter has been successfully cloned into E. coli cells. We are currently overexpressing RpoF and monitoring the effects of the downstream gene targets of this transcription factor on biofilm formation. To quantify the degree of up regulation of the downstream gene targets at the mRNA transcript level quantitative PCR (qPCR) is used.