Our team used PCR and LAMP amplification methods on E. coli K-12 cells as proof-of-concept for LAMP on pathogenic E. coli, Salmonella, Shigella, and Campylobacter cells. The first proof used PCR on extracted genomic DNA of non-pathogenic E. coli K-12, which validated the success of the designed PCR primers. PCR also verified that it is an inferior method for point of care (POC) DNA amplification compared to LAMP, as it required specific lab equipment like a thermocycler, user participation, and a great deal of time.

To gain an understanding of baseline amplified DNA concentration and the standard temperature of LAMP reactions, our team carried out LAMP on non-pathogenic E. coli using unmodified Bst polymerase. This round of LAMP reactions confirmed correct design of all 6 LAMP primers and acted as a base to which we could compare all future results of LAMP. We observed that E. coli RNA gave rise to a final DNA concentration of 3084.0 ng/µL via LAMP at 65°C. The results obtained from these experiments essentially acted as a control to compare the standard polymerase’s behaviour with that of our modified protein once we performed point mutations protein fusion, to observe whether our modifications would give rise to a positive effect. This also proved that at least one of our target pathogens could in fact be detected using the technology in our device, proving successful design of the LAMP-based test portion of the device.

In silico proof of concept was determined using YASARA and AlphaFold analysis. Point mutations K549W, K582L and Q584L were introduced to Bst polymerase in silico using YASARA software. This allowed us to determine the thermostability of these three residues before and after mutation by comparing Gibb’s free energy. We observed that all point mutations increased thermostability of the residue at that location, as well as the overall Bst polymerase. The aforementioned in silico proof is summarized in the following table, where ∆∆G represents Gibbs free energy, and more negative results indicate greater thermostability.

AlphaFold analysis was also performed to visualize 3D binding of Bst polymerase to Sac7e via a (GGGGS)4 flexible linker, as well as binding of the modified Bst to template DNA. This fusion therefore proved to further improve Bst polymerase in terms of processivity and strand displacing activity. This software confirmed that the length of the flexible linker was long enough to accommodate the distance of Sac7e to the DNA template.