Light-responsive building blocks for synthetic biology

合成生物学是一个新兴的研究领域，旨在模拟、修改或重新设计自然系统。其实施范围从用正交合成碱基扩展遗传密码、设计合成遗传电路和新陈代谢工程，到建造人造细胞和设计由修饰的天然成分合成的分子机器。这些努力中的一个关键因素是创造“生物艺术”，即健壮的、特征良好的模块，这些模块封装了特定的功能，可以根据已知的规则组合起来，设计出更复杂的组件。蛋白质和DNA及相关分子之间的相互作用支撑着合成生物学的许多目标，无论是目标核酸链用于编码基因，还是用作设计的结构或识别元件。因此，需要一种可以与其他模块结合以提供核酸酶或转录因子样行为的DNA结合生物工艺品。我们相信，光响应的DNA结合生物技术将在提供环境感知、指导或对合成生物结构的作用进行模式化或指导方面非常有用。我们的设计将基于细菌蛋白EL222的结构的解决方案，该蛋白仅在光激活状态下与DNA结合。EL222由一个光敏LOV结构域和一个C末端DNA结合域组成。LOV结构域存在于细菌、真菌和植物中的各种蓝光感应蛋白中。它们结合了FMN的一个分子，FMN充当感光发色团。这种分子对蓝光的吸收有利于FMN与蛋白质部分的一个氨基酸发生反应，形成一个共价光加合物，进而激活一个C末端的效应结构域。蓝光照射会释放C-末端结构域，然后该结构域可以与DNA结合。我们将在EL222和限制性内切酶FokI的催化区域之间设计一种杂交，FokI是一种众所周知的生物艺术，它已经连接到锌指蛋白和转录样效应域(TALE)，以创建高度特异的设计内切酶，从而创建一种可以在细胞中以高空间和时间分辨率激活的光响应核酸酶。我们将调整现有的定向进化试验，以提供一种工具来优化此类融合的有效性。随着进一步的工作，这种光核酸酶也将成为研究活细胞中基因功能的有价值的工具，并具有巨大的潜在临床实用价值，以空间定义的方式纠正人类疾病的突变，改善支撑健康的生物科学。

Synthetic biology is an emerging area of research that aims to mimic, modify or redesign natural systems. Its embodiments range from expanding the genetic code with orthogonal synthetic bases, designing synthetic genetic circuits, and metabolic engineering, to building artificial cells and designing synthetic molecular machines from modified natural components. A key element in a number of these endeavours is the creation of 'bioparts'; robust, well-characterized modules that encapsulate particular functions and can be combined by known rules to design a more complex component. Interactions between proteins and DNA and related molecules underpin many of the goals of synthetic biology, whether the target nucleic acid strand is used to encode genes or as a designed structural or recognition element. Therefore, a clear need exists for DNA-binding bioparts that can be combined with other modules to provide nuclease or transcription factor like behaviour. We believe a light-responsive DNA-binding biopart would be of great utility in providing environmental sensing, guiding or the ability to pattern or direct the action of synthetic biology constructs.We will base our design on the solution of the structure of the bacterial protein EL222, which binds to DNA only in its light activated state. EL222 consists of a light-sensitive LOV domain and a C-terminal DNA-binding domain. LOV domains are found in a variety of blue-light sensing proteins in bacteria, fungi and plants. They bind a molecule of FMN, which serves as the light-sensing chromophore. Absorption of blue light by this molecule is conducive to reaction between FMN and an amino acid of the protein part forming a covalent photo-adduct, which in turn activates a C-terminal effector domain. Irradiation with blue light releases the C-terminal domain that can then bind to DNA. We will design a hybrid between EL222 and the catalytic domain of the restriction endonuclease FokI, a well understood biopart which has been linked to zinc finger proteins and transcription like effector (TALE) domains to create highly specific designer endonucleases, thereby creating a photo-responsive nuclease that can be activated in cells with high spatial and temporal resolution. We will adapt an existing directed evolution assay to provide a tool to optimize the effectiveness of such fusions. With further work, such photonucleases will also be valuable tools to study the function of genes in live cells and have huge potential clinical utility to correct mutations in human disease in a spatially defined fashion improving biosciences underpinning health.