Directed Evolution of an Orthogonal Quadruplet Codon-based Genetic Code

用非标准氨基酸扩展遗传密码的策略提供了无与伦比的能力，使特定部位的赋予蛋白质具有新的功能。这使得研究蛋白质功能、结构、相互作用和定位的多种策略成为可能，例如通过编码生物物理探针或光笼残基。虽然已经产生了正交氨酰-tRNA合成酶/tRNA对，以结合结构不同的&gt；150氨基酸，但可以同时添加到遗传密码中的不同氨基酸的数量受到缺乏游离密码子的限制。最近，三个密码子被重新编码为同义词的大肠杆菌共生体的合成，使得直接获得23个氨基酸的遗传密码成为可能。我建议通过将三个空白的三联体密码子分成十二个四联体密码子来极大地扩展该大肠杆菌菌株的用途，这些密码子将由四联体tRNA解码。值得注意的是，这种方法消除了四倍体tRNAs与内源性tRNAs的竞争，并消除了蛋白质组有毒移码的风险。我将建立一种定向进化策略，以进化高度活跃的和特定的四胞体tRNA。随后，我将在重新编码的大肠杆菌菌株中组合几组相互正交的氨基酰-tRNA合成酶/四倍体tRNA对，建立一个长达32个氨基酸的遗传密码。接下来，我将利用这个浓缩的基于四重密码子的遗传密码来进化一个正交的核糖体，用于改进四重密码子但抑制三重密码子的解码，创建一个正交的四重密码子遗传密码。因此，DEQode将解锁四重密码子解码的潜力，提供前所未有的能力，将多个非规范氨基酸同时编码成蛋白质，并生成第一个独有的四重正交遗传密码。这个多学科的项目将利用我的蛋白质化学专业知识，同时为我提供广泛的合成生物学培训。

Strategies to expand the genetic code with non-canonical amino acids provide an unparalleled ability to site-specifically endowproteins with new functionalities. This has enabled a multitude of strategies to study protein function, structure, interactions, andlocalisation by, for example, encoding biophysical probes or photocaged residues. Although orthogonal aminoacyl-tRNA synthetase/tRNA pairs have been generated for the incorporation of >150 structurally diverse amino acids, the number of distinct amino acidsthat can be added to the genetic code simultaneously is restricted by a lack of free codons. Recently, the synthesis of an E. coligenome wherein three codons were recoded to synonyms has enabled straightforward access to a 23-amino acid genetic code. Ipropose to drastically expand the utility of this E. coli strain by splitting the three blank triplet codons into twelve quadruplet codonsto be decoded by quadruplet tRNAs. Significantly, this approach eliminates the competition of quadruplet tRNAs with endogenoustRNAs and removes the risk of toxic frameshifting of the proteome. I will establish a directed evolution strategy to evolve highly activeand specific quadruplet tRNAs. Subsequently, I will combine sets of mutually orthogonal aminoacyl-tRNA synthetase/quadruplettRNA pairs in the recoded E. coli strain, establishing an up-to-32-amino acid genetic code. Next, I will utilise this condensedquadruplet codon-based genetic code to evolve an orthogonal ribosome for improved quadruplet but suppressed triplet codondecoding, creating an orthogonal quadruplet codon genetic code. DEQode will thus unlock the potential of quadruplet codondecoding, provide unprecedented capacity to encode multiple non-canonical amino acids into proteins simultaneously, and generatethe first exclusively-quadruplet orthogonal genetic code. This multidisciplinary project will utilise my protein chemistry expertisewhile providing me with extensive synthetic biology training.