Investigating the functionality of the transmembranes in GusB by fusing it with LacY using the polymerase chain reaction.

Abstract

Glucuronides are produced during the detoxification pathway and removed via the uric pathway. The Escherichia coli living within the gut acquire these glucuronides for their survival by utilising the glucuronide transporter (GusB). GusB is encoded by the gusB gene which is located within the gus operon along with two other structural genes. However, the substrate binding sites of GusB are not currently known and therefore this research project was focused on fusing GusB with a different well-studied secondary transporter known as lactose ‘permease’ (LacY). LacY is a protein which facilitates the movement of lactose molecules across a membrane against the concentration gradient. This protein is been thoroughly studied and its substrate binding sites are known as well as which transmembranes they are located in. Hence, LacY’s structure was used for comparison with GusB as they share structural similarity i.e. 12 transmembranes. For this project, the first 6 membranes of LacY was fused with the last 6 transmembranes of GusB by undergoing fusion polymerase chain reaction (PCR). The PCR method required two steps in which fragments were fused together through overlap extension. Initially, the fusion was successful until a primer design error became evident during restriction digest. This resulted in the primers being redesigned and the PCR was repeated. However, obtaining the fusion since the correction proved difficult and required numerous troubleshoots in which various factors such as MgCl2 concentration, DNA concentration, temperature and extension time was altered. Due to this difficulty, the project did not progress further than PCR but has provided useful information for future troubleshooting and potential determination of the substrate binding sites.