Abstract:
A major purpose of this study is to isolate a heat shock promoter of groESL operon from Lactobacillus sp. by using polymerase chain reaction (PCR) and characterize its activity using a promoter probe vector pMU1327gfp. pMU1327g@, with Green Fluorescent Protein (GFP) as the reporter system, was constructed by insertion of a promoter-less gfp gene from plasmid pBCgfp into pMU1327, a shuttle vector for E.coli and Lactic Acid Bacteria. However, isolation of the groESL promoter using PCR is limited since there is no sequence upstream of the gi.oESL promoter available in the GenBank for primer design. Therefore, the promoter sequence of the groESL operon needs to be characterized. Chromosoinal DNA fragments containing the groESL operon then was identified by Southern Hybridization using a 967 bp homologous groEL probe generated by PCR. Southern blot analysis of Lactobacillus bulgaricus chromosoinal DNA cleaved with Hind111 produced 2 hybridizing bands of about 5.7 kb and 3.8 kb and a single band of about 2.9 kb when cleaved with KpnI. Several attempts were made to clone the fragments into pUC18 and the transformants were screened by either dot blot and colony hybridization analysis. No positive clone was detected in all experiments. However positive clones were further detected by PCR analysis. Unfortunately, all positive cloned could not survive in selective media. As a consequence, the fragment containing groESL operon could not be isolated. Inverse PCR then was utilized to characterize the sequences upstream and downstream of a 967 bp PCR product. No PCR product of expected size was obtained.