Engineering enzymes to enhance diagnostic devices for improved antibiotic stewardship

项目概述背景：Atlas寻求通过工程酶来开发改进的诊断系统，以增强Atlas的诊断技术。Atlas Genetics io系统是一个开创性的快速诊断平台，旨在检测衣原体和MRSA等传染病的存在，其中按需提供的快速，可操作的测试结果可以显着改善患者的结果，并更好地针对抗生素的使用，因此与抗菌素耐药性（AMR）主题相关。Atlas平台使用称为瓣状核酸内切酶的酶。它们是结构特异性DNA加工酶。Sayers实验室在开发这些酶和商业用途方面有着良好的记录（1），并在其表征，生产和修饰方面具有重要的专业知识（2，3）。这还将包括第二个学术导师，教授杰米霍布斯（物理系TUOS）谁将监督工作的原子力显微镜（ATM）成像的蛋白质DNA复合物。项目概要：这项合作旨在进一步表征Atlas使用的酶，并开发具有改进的信噪比特性的新的更强大的版本，并进行EE-DNA复合物的基本结构测定，就像我们对相关酶所做的那样（3）。Atlas Genetics Ltd Atlas拥有用于特定DNA电化学检测的微流体/硬件方面的专业知识，并正在开发用于快速检测病原体和遗传缺陷的新型即时检测设备。然而，重组核酸外切酶是该系统的核心，但Atlas在这一领域没有历史经验。在重组核酸外切酶技术、动力学分析和重组蛋白生产方面的互补专业知识存在于学术合作伙伴实验室中，他们开发了新的工程核酸外切酶（EE），非常适合Atlas平台。学生将表达、纯化和表征一个EE库，并评估它们在Atlas系统中与现有酶相比的性能。他/她将进行系统的分析开发，旨在提高信噪比、稳定性、检测限和特异性。我们有一个实时FRET为基础的溶液相分析，将用于产生定量生物物理数据，除了阿特拉斯分析。我们还将尝试与结合的DNA底物的EE的结晶和结构测定，以进一步告知蛋白质工程过程，并提供这些生物学和商业上重要的酶的新数据。1）Sayers，JR，Zhang J. Modified Exonucleases，2013，专利申请WO 2013079924 2）Wong，I. N.，塞耶斯，J.R.，& Sanders，C. M.（2016年）。噬菌体T5基因D10编码一个分枝迁移蛋白。科学报告，6.3）AlMalki，Faizah A.，等. Direct observation of DNA threading in flap endonuclease complex. Nature Structural & Molecular Biology（2016）.

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).