Engineering orthogonal split inteins as scalable tools for synthetic biology and biomanufacturing

合成生物学的一个重要目标是使用标准化的可互换部件来编程细胞行为的合成基因电路的合理设计和可预测实施。然而，迄今为止构建的大多数电路都是小规模的系统，这些系统是通过有限部件的昂贵且低效的“试错”方法构建的。这背后的一个残酷事实是，活细胞中复杂电路的工程目前受到缺乏可靠的布线方法以及良好表征和正交遗传调控构建模块的可用性的限制。此外，多功能的翻译后调控工具，如分裂内含肽代表了一个未开发/未充分开发的宝贵资源的合成生物学家。该项目旨在通过设计新型正交分裂内含肽介导的基因调控控制策略开发可扩展基因电路设计的新工具，包括设计模块化和正交遗传逻辑门和开关库，以显着扩展目前有限的遗传控制组件工具箱。该成果将通过直接解决该领域的关键瓶颈来改变基因网络工程的现状。我还将开发用于基因电路设计和诊断的新的高效自动化工具，以实现具有先进细胞信息处理能力的大规模遗传逻辑电路的快速，可预测的设计。这些工具有可能显着提高效率和规模，并将典型基因电路设计项目的开发时间从目前的几年缩短到几个月。此外，工程翻译后工具将被应用于开发突破性的生物技术应用，通过正交分裂内含肽介导的蛋白质连接，包括制造新型抗反射涂层生物材料和模块化多功能多特异性抗体，在该奖学金计划的后期阶段。成功的结果将为合成生物学带来变革性的使能技术，并在生物技术和治疗领域开辟广泛的创新蛋白质生物制造应用。

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardised interchangeable parts to program cellular behaviour. However, most of the circuits constructed so far are small-scale systems that have been constructed by costly and inefficient 'trial-and-error' methods with limited parts. A hard truth behind this is that the engineering of complex circuits in living cells is currently limited by the availability of well-characterised and orthogonal genetic regulatory building blocks in addition to the lack of robust wiring methods. Moreover, versatile post-translational regulation tools such as split inteins represent an untapped/underexplored invaluable resource for synthetic biologists. This project aims to develop new enabling tools for scalable gene circuit design via engineering novel orthogonal split intein mediated gene regulation control strategies, including engineering a library of modular and orthogonal genetic logic gates and switches to significantly expand the currently limited toolbox of genetic control components. The outcome will transform the present state of gene network engineering by directly addressing a key bottleneck in the field. I will also develop new efficient automation tools for gene circuit design and diagnosis to achieve rapid, predictable design of large-scale genetic logic circuits with advanced cellular information processing capacity. Such tools have the potential to significantly increase the efficiency and scale, and shorten development time of a typical gene circuit design project from the present several years to a few months. Further, the engineered post translational tools will be applied to develop groundbreaking biotechnology applications enabled by orthogonal split-intein mediated protein ligation including manufacturing novel anti-reflection coating biomaterials and modular versatile multipecific antibodies at a later stage of this fellowship program. The successful outcome will lead to a transformative enabling technology for synthetic biology and open up a wide range of innovative protein-based biomanufacturing applications in the biotechnology and therapeutic sectors.