Silaffin Tagged Polymerase for Nucleic Acid Amplification

Seevaratnam, Dushanth 

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There is an ever-growing demand for diagnostic relevant biological reagents. However, the high cost of these biomaterials limits their accessibility to burdened low- and middle-income countries. This thesis explores a synthetic biology approach to deliver a recombinant Geobacillus stearothermophilus (BST) DNA polymerase capable of low-cost purification, ease-of-use, and compatible with sensitive and specific loop mediated isothermal amplification (LAMP) assays. Fusing the enzyme with a silaffin tag (R5) and a fluorescent protein (mCherry) enabled rapid and cost-effective purification through unmodified silica immobilisation and built-in visual tracking from protein production to diagnostic assay, respectively. With the immobilised enzyme compatible with standard LAMP assays, additional elution steps were not necessary simplifying the purification from more traditional methods. The activity of the synthetic polymerase was highly dependent on the primer set for the target assay but had comparable outputs to commercial LAMP assays. In all 3 trialled sites (Cambridge, Ghana, Malaysia) the construct could be produced from a single flask culture (0.5L E. coli culture), at a rate of 2500 assays per 24 hours and achieve a limit of detection as low as 10 copies of target DNA. The synthetic polymerase was further evaluated with a Plasmodium falciparum diagnostics trial of 500 clinical venous blood samples, in collaboration with the West African Centre for Cell Biology of Infectious Pathogens. Achieving a maximum sensitivity and specificity of 69% and 95%, revealed some protocol discrepancies, but the recombinant protein showed promise with a faster turnaround time and better tolerance to sample contamination (blood) when compared to more standard nucleic acid amplification diagnostic methods. In addition, this thesis initiates consideration of an extremely thermostable red fluorescent protein through structural analysis of mCherry and thermal green protein (TGP) for future integration with other polymerases.

Hall, Elizabeth
Protein Engineering, Diagnostics, Loop-mediated isothermal amplification, Malaria
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Cambridge Trust, Commonwealth Scholarship