Light-responsive building blocks for synthetic biology

合成生物学是一个新兴的研究领域，旨在模仿，修改或重新设计自然系统。其实施方案的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代谢工程，到构建人工细胞和从修饰的天然成分设计合成分子机器。这些努力中的一个关键因素是创建“bioparts”;强大的，特征良好的模块，封装特定的功能，并可以通过已知的规则组合，以设计一个更复杂的组件。蛋白质和DNA以及相关分子之间的相互作用支撑了合成生物学的许多目标，无论靶核酸链是用于编码基因还是作为设计的结构或识别元件。因此，对于可以与其他模块组合以提供核酸酶或转录因子样行为的DNA结合生物部件存在明确的需求。我们相信光响应DNA结合生物部件在提供环境传感、引导或模式化或指导合成生物学构建体的作用的能力方面将具有很大的实用性。我们将基于细菌蛋白EL 222的结构的解决方案来设计我们的设计，该蛋白仅在其光激活状态下与DNA结合。EL 222由光敏LOV结构域和C-末端DNA结合结构域组成。LOV结构域存在于细菌、真菌和植物的多种蓝光传感蛋白中。它们与FMN分子结合，FMN分子作为光敏发色团。该分子吸收蓝光有助于FMN与蛋白质部分的氨基酸之间的反应，形成共价光加合物，这进而激活C-末端效应结构域。蓝光照射释放C-末端结构域，然后可以与DNA结合。我们将设计EL 222和限制性内切核酸酶FokI的催化结构域之间的杂交体，FokI是一种已被充分理解的生物部分，其已与锌指蛋白和转录样效应子（TALE）结构域连接以产生高度特异性的设计者内切核酸酶，从而产生可以在细胞中以高空间和时间分辨率激活的光响应性核酸酶。我们将调整现有的定向进化试验，以提供一种工具来优化这种融合的有效性。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用途，以空间定义的方式纠正人类疾病中的突变，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.