Effect of Plasmid Concentration on Natural Competence in Variovorax paradoxus MF295
Poster Number
37
Faculty Mentor Name
Paul Orwin
Research or Creativity Area
Natural Sciences
Abstract
Transformation efficiency in environmental bacteria is often limited by both physiological constraints and experimental design. This study tested how plasmid DNA concentration influences transformation competence in the rhizobacterium Variovorax paradoxus MF295. We hypothesized that higher plasmid doses would yield higher transformation frequencies.
To evaluate this, V. paradoxus MF295 cells were grown to mid‑log phase and concentrated to a uniform cell density. A kanamycin‑resistance plasmid was added directly to bacterial suspensions in microcentrifuge tubes at defined doses spanning low to high total DNA. Following incubation in rich medium, cell–DNA mixtures were diluted and plated onto non‑selective YE agar to quantify total viable cells and YE + kanamycin agar to enumerate transformants. Transformant colonies from low‑DNA treatments were subsequently re‑inoculated into YE broth containing kanamycin to enrich plasmid‑bearing cells for downstream plasmid isolation and confirmation.
Across replicates, detectable transformation occurred even at the lowest plasmid dose, while increases in DNA concentration produced only modest gains and in some cases plateaued, suggesting that transformation is constrained by cellular uptake or expression rather than DNA availability alone. These results highlight that environmental bacteria like Variovorax can acquire plasmids at relatively low extracellular DNA concentrations and that optimizing physiological conditions may be as important as increasing DNA dose when designing transformation protocols.
Location
University of the Pacific, DeRosa University Center
Start Date
24-4-2026 11:00 AM
End Date
24-4-2026 2:00 PM
Effect of Plasmid Concentration on Natural Competence in Variovorax paradoxus MF295
University of the Pacific, DeRosa University Center
Transformation efficiency in environmental bacteria is often limited by both physiological constraints and experimental design. This study tested how plasmid DNA concentration influences transformation competence in the rhizobacterium Variovorax paradoxus MF295. We hypothesized that higher plasmid doses would yield higher transformation frequencies.
To evaluate this, V. paradoxus MF295 cells were grown to mid‑log phase and concentrated to a uniform cell density. A kanamycin‑resistance plasmid was added directly to bacterial suspensions in microcentrifuge tubes at defined doses spanning low to high total DNA. Following incubation in rich medium, cell–DNA mixtures were diluted and plated onto non‑selective YE agar to quantify total viable cells and YE + kanamycin agar to enumerate transformants. Transformant colonies from low‑DNA treatments were subsequently re‑inoculated into YE broth containing kanamycin to enrich plasmid‑bearing cells for downstream plasmid isolation and confirmation.
Across replicates, detectable transformation occurred even at the lowest plasmid dose, while increases in DNA concentration produced only modest gains and in some cases plateaued, suggesting that transformation is constrained by cellular uptake or expression rather than DNA availability alone. These results highlight that environmental bacteria like Variovorax can acquire plasmids at relatively low extracellular DNA concentrations and that optimizing physiological conditions may be as important as increasing DNA dose when designing transformation protocols.
