Transformation, Expression and Activity Analysis of Recombinant Staphylococcus Autolysin in Bacillus
Enteric bacteria found in sewage water cause many diseases throughout the world. Through our project, we proposed a recombinant DNA method as a solution to this problem. Many of the commonly found sewage pathogens can be degraded by autolysin/cell wall hydrolases such as amidase and glucosaminidase. The Atl is the most predominant autolysin/peptidoglycan hydrolase in staphylococci. The AtlE of Staphylococcus epidermidisisbifunctional, composed of an amidasedomain and a glucosaminidase domain. We obtained the gene in a plasmid pTX15 with tetracycline resistance, secretion signal and transformed in Staphylococcus aureus which is a pathogenic bacteria. So we extracted the plasmid from it by alkaline lysis aided by cell wall hydrolase called lysostaphin. We transformed the plasmid by electroporation in Bacillus sp. which was screened as tetracycline sensitive. The transformed cells were selected with tetracycline but showed dramatic change in colony and cell morphology. So the taxonomic identity was checked with biochemical tests, followed by mass spectrometry (BrukerBiotyper MALDI- ToF). The supernatant from transformed culture significantly inhibited the growth of Gram positive S. aureus but not Gram negative Pseudomonas aeruginosa and E. coli. The secreted recombinant protein was analyzed by SDS- PAGE. The plasmid extracted from the transformed cells showed similar size by agarose gel electrophoresis. The dramatic effect of the transformation with atlE on Bacillus sp. needs to be further analyzed for better understanding.
Project work flow and pTX15 vector construct