Engineering enzymes to enhance diagnostic devices for improved antibiotic stewardship

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Project SummaryBackground: Atlas seeks to develop improved diagnostics systems by engineering enzymes to enhance Atlas' diagnostics technology. The Atlas Genetics io system is a pioneering rapid diagnostic platform designed to detect the presence of infectious diseases such as Chlamydia and MRSA, where a quick, actionable test result provided on-demand can significantly improve patient outcomes and better target the use of antibiotics and is thus of relevance to the Antimicrobial Resistance (AMR) theme. The Atlas platform uses enzymes known as flap endonucleases. These are structure-specific DNA processing enzymes. The Sayers lab has a track record in developing these enzymes and for commercial use (1) and significant expertise in their characterization, production and modification (2,3). This will also include a second academic supervisor, Prof. Jamie Hobbs (Physics Dept TUOS) who will supervise work on Atomic Force Microscopy (ATM) imaging of protein DNA complexes. Brief project outline: This collaboration seeks to further characterize the enzymes used by Atlas and to develop new more robust versions with improved signal-to-noise characteristics and to carry out basic structure determination of the EE-DNA complex as we have done for a related enzyme (3). Atlas Genetics Ltd Atlas has expertise in microfluidics/hardware for electrochemical detection of specific DNA and is developing new point-of-care devices for rapid detection of pathogens and genetic defects. However, a recombinant exonuclease is central to the system but Atlas has no historical expertise in this area. Complementary expertise in recombinant exonuclease technology, kinetic assays and recombinant protein production exists in the academic partner labs, who have developed new engineered exonucleases (EEs) that appear ideally suited to the Atlas platform.The student will express, purify and characterize a bank of EEs and evaluate their performance in the Atlas system compared with their current enzyme. He/she will carry out systematic assay development aimed at improving signal-to-noise ratios, stability, detection limits and specificity. We have a real-time FRET-based solution phase assay that will be used to produce quantitative biophysical data in addition to the Atlas assay. We will also attempt crystallization and structure determination of the EEs with bound DNA substrates to further inform the protein engineering process and provide new data on these biologically and commercially important enzymes. References from Sayers' lab relevant to project1) Sayers, JR, Zhang J. Modified Exonucleases, 2013, Patent application WO2013079924 2) Wong, I. N., Sayers, J. R., & Sanders, C. M. (2016). Bacteriophage T5 gene D10 encodes a branch-migration protein. Scientific Reports, 6.3) AlMalki, Faizah A., et al. Direct observation of DNA threading in flap endonuclease complexes. Nature Structural & Molecular Biology (2016).