The goal of this years project was to develop a point of care (POC) diagnostic device that can test for E. coli, Salmonella, Shigella and Campylobacter in drinking water. The mechanism that drives our test is an emerging nucleic acid amplification technique called “Loop Mediated Isothermal Amplification” or LAMP for short. LAMP requires 6 primers, so we performed a conserved motif analysis amongst over 400 different strains of our four target pathogens to target conserved 16s rRNA regions to develop a universal set of primers. To optimize LAMP, we engineered the polymerase responsible for the reaction; Bst polymerase. We based our design on modifications that had previously been done on Bst homologue Taq polymerase which is responsible for PCR reactions. Through the work of Xi, 2009 and Wang et. al, we were able to introduce three point mutations in the polymerase thumb domain and fusing the polymerase to DNA binding protien Sac7e to improve thermostability and processivity. Our lab work consisted of testing our LAMP primers and expressing our polymerase in an E. coli cell line.

Figure 1. Our lab work consisted of learning and performing LAMP as well as transformation, expression and purification of our fusion protein in E. coli.