Biophysical characterisation and design of optogenetic control elements

可诱导基因“开关”是合成生物学中的核心技术，其使得能够随意表达基因和编程细胞功能。光遗传学利用光控诱导剂，并允许对基因表达或抑制进行无与伦比的时间和空间控制。光遗传学的当前焦点是开发响应于不同波长的光的系统，潜在地允许单个细胞内的多个控制元件;例如，单独地响应于蓝光、绿色和红光。这种“多色控制”对于由工业合作伙伴（国家物理实验室; NPL）进行的生物计量学是特别期望的，因为当测量网络化生物系统时，靶向（时空）刺激提供更大的置信度。然而，直到最近，还缺乏绿色光诱导剂。发现并随后开发了一种含有辅酶B12依赖性CarH的结构，该结构对绿色光有反应，现在填补了这一空白。然而，这些CarH构建体（它们是嵌合融合蛋白）的体外生物物理表征是有限的，这限制了该系统在光遗传学中的改进和扩展。该项目将使用一系列生物物理技术来研究CarH诱导剂的机制。该项目将利用（光激活）时间分辨光谱学-基于光学和质谱法-来研究辅酶B12的结合以及蛋白质低聚状态和与DNA结合的光依赖性，这在功能上是相关的。新嵌合蛋白的诱变和构建将用于研究机制和开发具有改善或改变的活性的系统。然后如前所述，使用绿色光测试这些新变体的体内优化光遗传学转录调控。然后，它们将被整合到已建立的多色光遗传致动器系统中。该项目涉及最近开发的基于天然辅酶B12依赖蛋白的光遗传学控制系统的生物物理特性。它福尔斯“技术和方法发展”，“合成生物学”和更广泛的“世界级生物科学”的职权范围，并牢固地嵌入化学，生物学和物理学的界面，这是BBSRC在“探索新的工作方式”议程中的关键驱动力。它借鉴了数学（数据分析）的核心生物科学技能，并提供了先进的研究培训，重组蛋白生产和生物物理表征使用一系列的溶液光谱和质谱技术。总的来说，这项工作将提供一个高度跨学科的方法（生物）化学/生物制药为基础的研究，提供高度多样化的培训机会，博士生。曼彻斯特的主管与国家物理实验室（NPL）的工业合作伙伴建立了长期的合作关系，学生将获得能够每天访问当地主管实验室的额外好处，因为它们位于同一栋建筑物内，曼彻斯特生物技术研究所（MIB）。

Inducible gene 'switches' are a core technology in synthetic biology that enable at-will gene expression and the programming of cellular function. Optogenetics makes use of light-controlled inducers and allows unparalleled temporal and spatial control of either gene expression or repression. A current focus of optogenetics is the development of systems that respond to different wavelengths of light, potentially allowing multiple control elements within a single cell; e.g. individually responding to blue, green and red light. Such 'multichromic control' is particularly desirable to the biological metrology conducted by the industrial partner (National Physical Laboratory; NPL) because targeted (spatiotemporal) stimulation provides greater confidence when measuring networked biological systems. However, until recently, green light inducers were lacking. The discovery and subsequent development of a construct containing coenzyme B12-dependent CarH, which responds to green light, now plugs this gap. However, the in vitro biophysical characterisation of these CarH constructs (they are chimeric fusion proteins) is limited, which limits the improvement and extension of this system in optogenetics. This project will use a range of biophysical techniques to investigate the mechanism of CarH inducers. The project will make use of (light-activated) time-resolved spectroscopy - both optical and mass spectrometry based - to characterise coenzyme B12 binding and the light-dependence of both the protein oligomeric state and binding to DNA, which are functionally relevant. Mutagenesis and construction of new chimeric proteins will be used to both investigate mechanism and develop systems with improved or altered activity. These new variants will then be tested for optimised optogenetic transcriptional regulation in vivo using green light as previously described. They will then be integrated, into established multichromic optogenetic actuator systems. The project involves the biophysical characterisation of a recently developed optogenetic control system, based on a natural coenzyme-B12 dependent protein. It falls within the remit of 'technologies and methodological development', 'synthetic biology', and more generally 'world class bioscience' and is firmly embedded at the interface of chemistry, biology and physics, a key driver for BBSRC in the 'Exploiting new ways of working' agenda. It draws on the core bioscience skills of mathematics (data analysis), and provides advanced research training in recombinant protein production and biophysical characterisation using a range of solution spectroscopy and mass spectrometry techniques. Overall, the work will provide a highly interdisciplinary approach to (bio)chemistry/ biophysics-based research, offering highly diverse training opportunities to a PhD student. The Manchester-based supervisors have long-established collaborations with the industrial partner at the National Physical Laboratory (NPL) and the student will have the additional benefit of being able to access the local supervisors' laboratories on a daily basis, as they are co-located within the same building, the Manchester Institute of Biotechnology (MIB).