GFP expression and phenotypic variation in recombinant Variovorax paradoxus EPS
Poster Number
10A
Research or Creativity Area
Natural Sciences
Abstract
Variovorax paradoxus EPS is a Gram-negative aerobic bacterium that is commonly found in soil. The vector pBBR-8k has been previously introduced in V. paradoxus EPS, containing a Green Fluorescence Protein (GFP) under the control of the arabinose promoter (pBAD). Our initial goal was to determine the nature and efficacy of this induction, and to screen for mutants that had stronger arabinose control of gene expression. We observed in our initial experiments that we had strong correlation between GFP expression control and colony morphology in our control (unmutated) population. This led to an extension of the experimental approach to isolate strongly fluorescent colonies under arabinose induction with different morphologies, and to use these isolates to test the regulation of fluorescence in V. paradoxus EPS and the role of colony morphology in that regulation.
We inoculated E. coli + pBBR-8k, V. paradoxus + pBBR-8k, and transposon library samples transformed with pBBR-8k were plated onto both freshwater/glucose media and freshwater/arabinose media containing kanamycin. We observed modest variation in GFP expression in both the transposon library and the V. paradoxus EPS control. Since we did not see variation only associated with the transposon library we focused on the behavior of the control sample. Two different colony phenotypes were identified, one that had a dense colony and strong GFP induction on the plate, and others that were “goopy” and not as uniformly shaped as the others. These “goopy” colonies showed less robust GFP induction. To start, the bacteria were plated onto glucose and kanamycin plates, then were replated onto arabinose and kanamycin plates. Colonies and individual cells were imaged using the ECHO Revolve epi-fluorescence microscope to detect GFP expression.
Location
Don and Karen DeRosa University Center (DUC) Poster Hall
Start Date
27-4-2024 10:30 AM
End Date
27-4-2024 12:30 PM
GFP expression and phenotypic variation in recombinant Variovorax paradoxus EPS
Don and Karen DeRosa University Center (DUC) Poster Hall
Variovorax paradoxus EPS is a Gram-negative aerobic bacterium that is commonly found in soil. The vector pBBR-8k has been previously introduced in V. paradoxus EPS, containing a Green Fluorescence Protein (GFP) under the control of the arabinose promoter (pBAD). Our initial goal was to determine the nature and efficacy of this induction, and to screen for mutants that had stronger arabinose control of gene expression. We observed in our initial experiments that we had strong correlation between GFP expression control and colony morphology in our control (unmutated) population. This led to an extension of the experimental approach to isolate strongly fluorescent colonies under arabinose induction with different morphologies, and to use these isolates to test the regulation of fluorescence in V. paradoxus EPS and the role of colony morphology in that regulation.
We inoculated E. coli + pBBR-8k, V. paradoxus + pBBR-8k, and transposon library samples transformed with pBBR-8k were plated onto both freshwater/glucose media and freshwater/arabinose media containing kanamycin. We observed modest variation in GFP expression in both the transposon library and the V. paradoxus EPS control. Since we did not see variation only associated with the transposon library we focused on the behavior of the control sample. Two different colony phenotypes were identified, one that had a dense colony and strong GFP induction on the plate, and others that were “goopy” and not as uniformly shaped as the others. These “goopy” colonies showed less robust GFP induction. To start, the bacteria were plated onto glucose and kanamycin plates, then were replated onto arabinose and kanamycin plates. Colonies and individual cells were imaged using the ECHO Revolve epi-fluorescence microscope to detect GFP expression.
