Sense Codon Reassignment: Evaluating the Plasticity of the E. Coli Genetic Code

该项目由数学和物理科学局化学部的生命过程化学计划和生物科学局分子和细胞生物科学部的系统和合成生物学集群共同资助。蛋白质是生命系统中的中心角色，在细胞中执行绝大多数催化、信号、结构和控制功能。蛋白质的化学成分在很大程度上由蛋白质的20个基因编码的氨基酸组成单元的化学组成。虽然这组天然氨基酸对生命系统来说足够了，但它编码的化学功能有限。基因可编码化学功能的扩展超过了20种天然氨基酸所能提供的功能，将为探索和开发蛋白质序列和功能空间开辟广阔的新前景。通过遗传密码扩展来扩大蛋白质的可编码化学是一种使能技术，它提供了极大地增加蛋白质功能的潜力。这项研究将探索大肠杆菌中有义密码子重新分配的空间，以确定一组可重新分配的密码子用于遗传密码扩展。这项研究将导致改进的非规范氨基酸掺入技术，允许多个非规范氨基酸的多个副本被掺入任何感兴趣的蛋白质中。这些实验的追求将有助于培养下一代跨学科的科学家和工程师，为强大的具有竞争力的STEM劳动力做出贡献。以tRNA/氨基酰tRNA合成酶系统的形式构建的针对正义密码子重新分配进行优化的遗留基础设施将为蛋白质科学研究社区提供新的工具，并通过快速提高基于蛋白质的材料和设备的生成速度直接造福社会。在这个奖项下，菲斯克和他的研究团队将研究通过正义密码子重新分配来扩展遗传密码。该研究项目将使用基于荧光的筛选来测量在多大程度上可以使用最常用于无意义抑制的正交tRNA/AARS对来重新分配在大肠杆菌中通过摆动相互作用自然读取的正义密码子。相同的荧光屏幕将进一步用于进化tRNA/AARS系统，以更好地解码重新分配的正义密码子，评估氨基酰tRNA合成酶-tRNA识别结构域的可塑性，并探索翻译机制可以在多大程度上被塑造以适应扩展的遗传密码。被识别和优化以重新分配正义密码子的系统将被组合，以产生包含22和23个氨基酸的遗传密码。该项目的联合实验目标提供了一系列新的测量方法，以更好地定位大肠杆菌中翻译系统的可塑性，并评估具有代表性的tRNA/AARS对可以被修改的程度，以重新分配正义密码子的含义。

This project is jointly funded by the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences and the Systems and Synthetic Biology Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences. Proteins are the central actors in living systems, performing the vast majority of catalytic, signaling, structural, and controlling functions in cells. The chemistry of proteins is largely determined by the chemistry of the 20 genetically-encoded amino acid building blocks of proteins. Although sufficient for living systems, this set of natural amino acids encodes a limited set of chemical functionalities. The expansion of the genetically encodable chemical functionality beyond that afforded by the 20 natural amino acids will open vast new vistas of protein sequence and functional space for exploration and exploitation. Enlarging the encodable chemistry of proteins through genetic code expansion is an enabling technology that offers the potential to dramatically increase the functions of proteins. This research will explore the space of sense codon reassignment in E. coli to identify a set of reassignable codons for genetic code expansion. The research will lead to improved technologies for non-canonical amino acid incorporation that allow multiple copies of multiple non-canonical amino acids to be incorporated into any protein of interest. The pursuit of these experiments will help prepare of the next generation of interdisciplinary scientists and engineers to contribute to a strong competitive STEM workforce. The legacy infrastructure built in the form of tRNA/aminoacyl tRNA synthetase systems optimized for sense codon reassignment will provide new tools to the protein science research community and direct benefits to society through rapid increases in the rate of generation of improved protein-based materials and devices.Under this award, Fisk and his research team will study the expansion of the genetic code through sense codon reassignment. The research project will employ a fluorescence-based screen to measure the extent to which sense codons that naturally read through wobble interactions in E. coli can be reassigned using the orthogonal tRNA/aaRS pairs most commonly employed for nonsense suppression. The same fluorescence screen will be further employed to evolve the tRNA/aaRS systems to better decode reassigned sense codons, to evaluate the plasticity of aminoacyl tRNA synthetase-tRNA recognition domains, and explore the extent to which the machinery of translation can be molded to accommodate expanded genetic codes. The systems identified and optimized to reassign sense codons will be combined to generate genetic codes containing 22 and 23 amino acids. The combined experimental aims of the project provide a host of new measurements to better map the plasticity of the translational system in E. coli and evaluate the degree to which representative tRNA/aaRS pairs can be modified to reassign the meaning of sense codons.