Construction of recombinant plasmids to study genes of the bph cluster.

Burkholderia xenovarans (strain LB400) is a bacterium found in soil that has the ability to degrade polychlorinated biphenyls and other contaminants. The bacterium's ability to catabolize biphenyls is directly related to the presence of a locus of approximately thirteen genes known as the bph cluster. LS2 cells are LB400 cells that lack the bph cluster. In order to understand individual genes of the bph cluster, portions of this cluster can be introduced into LS2 cells and will provide important information on the function of these genes, along with their interactions with other genes in the cluster. A recombinant plasmid was created by digesting pKT230 and inserting another plasmid called pGEM4A2 (pUC18 + orf0-bphA). This new plasmid was called pKT230.1. This hybrid plasmid was introduced into E. coli cells via transformation. The orientation of the insert was determined. We will introduce this plasmid into LS2 cells by electroporation and selection on kanamycin. In addition, we will attempt to remove unnecessary portions of pKT230.1 and introduce this new plasmid into LS2 cells. LS2 cells with these recombinant plasmids will be tested to detect and analyze expression of bphA.