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Evaluating Expression Control Systems in Variovorax paradoxus EPS using GFP Expression

2023
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Poster Description

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.

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