Biophysical characterisation and design of optogenetic control elements

诱导基因“开关”是合成生物学中的一项核心技术，可以实现基因的随意表达和细胞功能的编程。光遗传学利用光控诱导剂，允许对基因表达或抑制进行无与伦比的时间和空间控制。光遗传学目前的一个重点是开发响应不同波长光的系统，可能在单个细胞内允许多个控制元件；例如，分别对蓝、绿、红灯作出反应。这种“多色控制”对于工业合作伙伴（国家物理实验室；NPL）进行的生物计量学特别理想，因为目标（时空）刺激在测量网络生物系统时提供了更大的信心。然而，直到最近，还缺乏绿光诱导器。一种含有辅酶b12依赖性CarH的结构的发现和随后的发展，这种结构对绿光有反应，现在填补了这一空白。然而，这些CarH结构体（它们是嵌合融合蛋白）的体外生物物理特性是有限的，这限制了该系统在光遗传学中的改进和扩展。本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（光激活的）时间分辨光谱——基于光学和质谱——来表征辅酶B12的结合，以及蛋白质寡聚态和与DNA结合的光依赖性，这些都是功能相关的。诱变和新嵌合蛋白的构建将用于研究机制和开发具有改进或改变活性的系统。如前所述，这些新变异将在体内使用绿光进行优化的光遗传转录调节测试。然后将它们集成到已建立的多色光致动器系统中。该项目涉及最近开发的光遗传控制系统的生物物理特性，该系统基于天然辅酶- b12依赖蛋白。它属于“技术和方法发展”、“合成生物学”以及更普遍的“世界级生物科学”的范畴，并牢固地嵌入化学、生物学和物理学的界面，是BBSRC“探索新的工作方式”议程的关键驱动因素。它利用数学（数据分析）的核心生物科学技能，并使用一系列溶液光谱和质谱技术提供重组蛋白生产和生物物理表征方面的高级研究培训。总的来说，这项工作将为（生物）化学/生物物理学的研究提供高度跨学科的方法，为博士生提供高度多样化的培训机会。曼彻斯特的导师与国家物理实验室（NPL）的工业合作伙伴建立了长期的合作关系，学生将获得额外的好处，即能够每天访问当地导师的实验室，因为它们位于同一栋建筑内，即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).