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，旨在检测衣原体和耐甲氧西林金黄色葡萄球菌等传染病的存在，按需提供快速、可操作的检测结果可以显著改善患者的预后，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平台使用了被称为翻盖核酸内切酶的酶。这些都是结构特定的DNA处理酶。塞耶斯实验室在开发这些酶和用于商业用途方面有着良好的记录(1)，并在它们的表征、生产和修饰方面拥有丰富的专业知识(2，3)。这还将包括第二位学术导师，Jamie Hobbs教授(物理系TUOS)，他将监督蛋白质DNA复合体的原子力显微镜(ATM)成像工作。项目简介：这项合作旨在进一步确定Atlas使用的酶的特性，开发具有改进的信噪比特性的更强大的新版本，并像我们对相关酶所做的那样，对EE-DNA复合体进行基本结构测定(3)。Atlas Genetics Ltd Atlas拥有用于电化学检测特定DNA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的护理点设备。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。学术伙伴实验室在重组核酸外切酶技术、动力学分析和重组蛋白质生产方面存在互补的专业知识，他们开发了新的工程核酸外切酶(EES)，似乎非常适合Atlas平台。学生将表达、纯化和鉴定一组EES，并评估它们在Atlas系统中与当前酶的性能。他/她将进行系统的分析开发，旨在提高信噪比、稳定性、检测下限和特异性。我们有一种基于FRET的实时溶液相分析，除了Atlas分析外，它还将用于产生定量的生物物理数据。我们还将尝试结合DNA底物的EES的结晶和结构测定，以进一步了解蛋白质工程过程，并提供关于这些具有生物和商业重要性的酶的新数据。来自Sayers实验室的与项目相关的参考文献1)Sayers，Jr，Zhang J.修改的核酸外切酶，2013，专利申请WO2013079924 2)Wong，I.N.，Sayers，J.R.，&Sanders，C.M.(2016)。噬菌体T5基因D10编码一个分支迁移蛋白。科学报告，阿尔马尔基、法伊扎·A等人。直接观察DNA在瓣状核酸内切酶复合体中的穿线。《自然结构与分子生物学》(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).