Evaluating Expression Control Systems in Variovorax paradoxus EPS using GFP Expression

Poster Number

8B

Lead Author Major

Pre-Dentistry

Lead Author Status

Sophomore

Second Author Major

Pre-Dentistry

Second Author Status

Junior

Format

Poster Presentation

Faculty Mentor Name

Paul Orwin

Faculty Mentor Department

Biological Sciences

Graduate Student Mentor Name

Rebecca Rafique

Abstract/Artist Statement

Variovorax paradoxus is an aerobic, gram-negative bacterium frequently found in soil communities. The Variovorax paradoxus EPS strain was isolated from the sunflower rhizosphere and has been shown to have a role as a plant growth promoting rhizobacteria (PGPR). Other V. paradoxus strains are also known to have biodegradative abilities due to their conjugative pesticide-degrading plasmids. To evaluate conjugation between Variovorax strain in the lab, we need it to express the green fluorescent protein (GFP), and we want to tightly regulate this expression so that we can use this vector in future experiments. Two different vectors using arabinose (pBBR8k) and anhydrotetracycline (pBBR2k) were previously introduced into V. paradoxus EPS using electroporation. Two experiments to evaluate the level of gene expression control were performed. In the first experiment, we focused on attempting to control the expression of GFP in Variovorax using both of the specified inducers. In the second experiment, high concentrations of only one specific inducer–anhydrotetracycline– were used. After induction, Variovorax paradoxus EPS was observed under a fluorescent microscope and compared to Escherichia coli with the same GFP plasmids. Underneath the microscope, the induced E. coli glowed bright green while the uninduced culture did not. The recombinant V. paradoxus EPS showed weak fluorescence regardless of inducer concentration. GFP protein was also assessed using SDS-PAGE and Western Blotting to analyze protein levels. Our experiment led us to conclude that arabinose or anhydrotetracycline promoters could not be used to control the GFP expression in Variovorax.

Location

Information Commons, William Knox Holt Memorial Library and Learning Center

Start Date

29-4-2023 10:00 AM

End Date

29-4-2023 1:00 PM

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Apr 29th, 10:00 AM Apr 29th, 1:00 PM

Evaluating Expression Control Systems in Variovorax paradoxus EPS using GFP Expression

Information Commons, William Knox Holt Memorial Library and Learning Center

Variovorax paradoxus is an aerobic, gram-negative bacterium frequently found in soil communities. The Variovorax paradoxus EPS strain was isolated from the sunflower rhizosphere and has been shown to have a role as a plant growth promoting rhizobacteria (PGPR). Other V. paradoxus strains are also known to have biodegradative abilities due to their conjugative pesticide-degrading plasmids. To evaluate conjugation between Variovorax strain in the lab, we need it to express the green fluorescent protein (GFP), and we want to tightly regulate this expression so that we can use this vector in future experiments. Two different vectors using arabinose (pBBR8k) and anhydrotetracycline (pBBR2k) were previously introduced into V. paradoxus EPS using electroporation. Two experiments to evaluate the level of gene expression control were performed. In the first experiment, we focused on attempting to control the expression of GFP in Variovorax using both of the specified inducers. In the second experiment, high concentrations of only one specific inducer–anhydrotetracycline– were used. After induction, Variovorax paradoxus EPS was observed under a fluorescent microscope and compared to Escherichia coli with the same GFP plasmids. Underneath the microscope, the induced E. coli glowed bright green while the uninduced culture did not. The recombinant V. paradoxus EPS showed weak fluorescence regardless of inducer concentration. GFP protein was also assessed using SDS-PAGE and Western Blotting to analyze protein levels. Our experiment led us to conclude that arabinose or anhydrotetracycline promoters could not be used to control the GFP expression in Variovorax.