Expanding the functions of a 57 codon recoded E.coli genome

所有基因组中都嵌入了一种基本且高度保守的密码，是生物生命的基础。这个密码被称为“标准遗传密码”，它定义了遗传信息如何在生物制造过程中将氨基酸构建块转化为蛋白质。利用标准遗传密码的普遍性已经彻底改变了材料、食品、化学和能源工业。尽管在少数生物体中存在与标准遗传密码的微小偏差，但标准遗传密码经受住了近35亿年的遗传漂变，在生命的所有领域都高度保守。遗传密码子是遗传物质（DNA或RNA）的三联体单位，可以定义蛋白质构建块。64个三重密码子映射到20个标准氨基酸（蛋白质的构建块），导致相同的氨基酸被多个同义密码子编码。通过在活细胞中合理地构建合成基因组，使用一组减少的同义密码子，释放的密码子可以重新分配，将非天然氨基酸整合到蛋白质中。通过系统的密码子替换，研究人员正在朝着计算设计的细菌基因组的最终组装前进，该基因组依赖于57个密码子，而不是通用的64个密码子。本项目将探索完整组装的57密码子合成细菌细胞，并对其进行进一步修饰，以实现安全的生物围护和遗传隔离，防止遗传信息与环境交换。研究人员还将建立一个开放访问的数据库，使非规范氨基酸结合的简化计算方法成为可能。作为一种拓展形式，研究人员将组织为期两天的开放式网络研讨会系列，每年与全球从事合成细胞工程项目的哲学家，早期职业研究人员，STEM研究生和科学专家一起讨论与合成细胞相关的未来前景和伦理问题。标准遗传密码将基因组中的64个三联体密码子与20个典型氨基酸相结合。将标准遗传密码编码的信息进一步翻译为蛋白质涉及到适配tRNA分子、核糖体和氨基酰基tRNA合成酶。研究人员正在通过替换编码典型氨基酸丝氨酸、亮氨酸、丙氨酸和琥珀色终止密码子的7个密码子来组装一个含有57个密码子的大肠杆菌基因组。释放的密码子将包含多个非规范氨基酸，并为工程遗传信息建立生物防护。研究人员将通过研究蛋白质组对长期非规范氨基酸依赖的适应性，获得关于细胞可塑性的基本见解。该研究还将通过跟踪密码子捕获和模糊解码等过程，为遗传密码进化提供生物学见解，这些过程发生在重新编码的基因组对非规范氨基酸的长期适应和依赖过程中。该项目还将建立一个开放获取的遗传密码扩展数据库，并提供可用的计算工具，以促进对非规范氨基酸的研究。总之，预计该项目将为与遗传密码和合成细胞工程相关的学术和工业努力提供新的工具和知识，同时促进合成生物系统的安全使用，并将这些方面传达给广泛的研究人员和STEM学生。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

A fundamental and highly conserved code is embedded in all genomes and underlies biological life. This code is known as the ‘standard genetic code’, which defines how genetic information translates amino acid building blocks into proteins during their biological manufacturing process. Harnessing the universality of the standard genetic code has revolutionized the material, food, chemical, and energy industry. Although minor deviations from the standard code exist in a few organisms, the standard genetic code has withstood nearly 3.5 billion years of genetic drift by being highly conserved across all domains of life. The genetic codon is a triplet unit of a genetic material (DNA or RNA) that can define a protein building block. The mapping of 64 triplet codons to the 20 canonical amino acids, the building blocks of proteins, results in the same amino acid being encoded by multiple synonymous codons. By rationally building synthetic genomes in living cells, with a reduced set of synonymous codons, the liberated codons can be reassigned to incorporate non-natural amino acids into proteins. By systematic codon replacement, researchers are progressing towards the final assembly of a computationally designed bacterial genome that relies on 57 codons instead of the universal 64 codons. In this project, the fully assembled 57-codon synthetic bacteria cell will be explored and further modified for safe bio-containment and genetic isolation to prevent exchange of genetic information with the environment. The researchers will also build an open-access database to enable streamlined computational approaches for non-canonical amino acid incorporation. As a form of outreach, the researchers will organize a two-day open webinar series annually with philosophers, early-career researchers, STEM graduate students, and scientific experts engaged in synthetic cell engineering projects globally to discuss future perspectives and ethical issues associated with synthetic cells.The standard genetic code maps the 64 triplet codons in the genome with the 20 canonical amino acids. Translating the information encoded in the standard genetic code to proteins further involves adaptor tRNA molecules, ribosomes, and amino-acyl tRNA synthetases. Researchers are progressing towards assembling a 57-codon Escherichia coli genome by replacing seven codons that encode the canonical amino acids serine, leucine, alanine and the amber stop codon with their synonymous alternatives. The liberated codons will incorporate multiple non-canonical amino acids and establish biocontainment for engineered genetic information. The researchers will gain fundamental insights on cellular plasticity by studying the adaptations of the proteome towards long-term non-canonical amino acid dependence. The study will also provide biological insights into genetic code evolution by tracking for processes such as codon-capture and ambiguous decoding during the long-term adaptation and dependence of the recoded genome to non-canonical amino acids. The project will also build an open-access database for genetic code expansion and make available computational tools to facilitate the research with non-canonical amino acids. In sum, it is expected that this project will provide new tools and knowledge for academic and industrial efforts related to genetic code and synthetic cell engineering while simultaneously facilitating the safe use of synthetic biological systems and communicating these aspects to a wide range of researchers and STEM students.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.