Light-responsive building blocks for synthetic biology

合成生物学的光响应构建模块

• 批准号: BB/M006158/1
• 负责人: Rudolf Allemann
• 金额: $ 57.93万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM006158%2F1
• 关键词: Light; responsive; building; blocks; synthetic

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.

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。它的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代谢工程，到构建人工细胞和设计由改良的自然成分合成的合成分子机器。其中一些努力的一个关键要素是创造“生物制品”；封装特定功能的健壮的、具有良好特征的模块，可以通过已知规则组合以设计更复杂的组件。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，无论目标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dna结合生物部件的明确需求是存在的，这种生物部件可以与其他模块结合以提供类似核酸酶或转录因子的行为。我们相信光响应dna结合生物技术在提供环境感知、指导或模式或指导合成生物学结构的行动能力方面将具有很大的实用性。我们的设计将基于细菌蛋白EL222的结构溶液，它只在光激活状态下与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感应蛋白中。它们结合一个FMN分子，充当感光发色团。该分子对蓝光的吸收有利于FMN与蛋白质部分的氨基酸之间的反应，形成共价光加合物，进而激活c端效应域。蓝光照射释放c端结构域，然后与DNA结合。我们将在EL222和限制性内切酶FokI的催化结构域之间设计一种杂交，FokI是一种众所周知的生物艺术，与锌指蛋白和转录样效应（TALE）结构域相关联，以创建高度特异性的设计内切酶，从而创建一种光响应核酸酶，可以在高空间和时间分辨率的细胞中激活。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，这些光子核酸酶也将成为研究活细胞中基因功能的宝贵工具，并在以空间定义的方式纠正人类疾病突变方面具有巨大的临床应用潜力，从而改善支撑健康的生物科学。

项目成果

Spatio-Temporal Photoactivation of Cytotoxic Proteins.

• DOI: 10.1002/cbic.202200115
• 发表时间: 2022-06-20
• 期刊: CHEMBIOCHEM
• 影响因子: 3.2
• 作者: Cruz-Samperio, Raquel;Mart, Robert J.;Luk, Louis Y. P.;Tsai, Yu-Hsuan;Jones, Arwyn T.;Allemann, Rudolf K.
• 通讯作者: Allemann, Rudolf K.