Evaluation of a pBAD based promoter for control of gene expression in Variovorax paradoxus

Toxin-antitoxin (TA) systems are widespread genetic elements in bacteria thought to contribute to many phenotypes. They function by using differential protein stability to control cell lysis during growth. This project is an effort to evaluate a tightly regulated gene expression system using arabinose as an inducer to study the role of TA systems in strains of Variovorax paradoxus, an important soil bacterium. These preliminary studies using a GFPuv reporter assay will provide the necessary foundation for designing our future experiments to evaluate these functional elements in phenotypes such as biofilm formation and horizontal gene transfer. Our project goal was to evaluate the utility of a broad host range vector that uses arabinose to control expression of GFPuv. This plasmid, pBBR8k-GFPuv, was isolated from Escherichia coli and transformed into several strains of Variovorax paradoxus using electroporation. Transformants were isolated and the plasmid was verified by agarose gel electrophoresis. Reporter gene expression was verified using fluorescence microscopy on cultures at several time points after induction, and SDS-PAGE was also employed to evaluate the expression levels. The expression profile was compared to similar experiments in the original E. coli strain. Fluorescence microscopy on the original E. coli strain showed clearly that arabinose could effectively induce GFPuv based fluorescence, but the SDS-PAGE data was much more ambiguous. The plasmids were successfully transformed into Variovorax paradoxus, but the induction experiments suggest that either the regulation of the gene is not as tight, or the induction parameters are different. This will be the basis for future experimentatio