Enabling synthetic biology with an expanded library of engineered orthogonal genetic logic gates and switches

合成生物学的一个重要目标是合理设计和可预测地实现合成基因电路，使用标准化和可互换的部件来编程细胞行为。然而，与电子数字电路不同的是，生物电路中的元件不是由物理绝缘的电线连接的，生物信息的流动必须依靠它们特定的化学相互作用来避免串扰。因此，相同的遗传部分可能不会在一个综合系统中使用两次，以防止它们之间潜在的意外相互作用。因此，对于包含多个元件的大型基因电路的兼容性和可扩展性设计来说，正交的部件和模块是必要的。正交性意味着新添加的部分和模块不应该与那些存在于工程生物系统以及宿主遗传背景中的部分和模块串扰。到目前为止，大多数构建的基因电路都是小规模的系统，这些系统是通过昂贵而低效的“试错”方法构建的，部件非常有限。例如，从第一个3基因切换开关到迄今为止在单个细胞中构建的最大的11基因4输入与逻辑门，已经花了将近12年的时间。这种缓慢背后的一个残酷的事实是，活细胞中复杂电路的工程设计目前受到充分表征和正交（非串扰）基因调控构建块的可用性的限制。因此，合成生物学的一个迫切需要是用许多功能正交的元素来扩展目前有限的生物部件工具箱，以扩大我们构建大型复杂电路的能力。然而，扩大合成生物学工具箱中可用正交成分的范围仍然是一个巨大的基础挑战。该项目旨在通过开发两种新颖的可扩展工具来解决这一挑战，以设计一个扩展的多用途正交遗传构建块库。特别是，我们将构建模块化和正交遗传NAND和NOR逻辑门库；这些是通用逻辑门，它们的组合可以用来完成任何任意复杂的布尔逻辑运算，为细胞过程控制提供了一种强大的可扩展方法。此外，我们将从这些工程逻辑门的不同排列中创建多层遗传程序，以证明在单个细胞中组成高阶信号处理和转录控制功能的潜力。例如，工程遗传程序将用于实现高级逻辑计算设备- 1位全加法器，该加法器以指定的逻辑方式接收三个化学输入以产生两个光输出。此外，我们将证明大型复杂的转录控制程序可以在微生物细胞工厂中实施，以精确和快速地调整高价值化学物质（紫罗兰素）生物合成途径中的基因表达谱。本研究的工程可扩展工具将显著增加合成生物学有限工具箱中的正交控制元件、门和线的数量，从而实现可编程细胞高级行为的大规模复杂遗传控制程序。这一成功的结果将导致许多生物技术行业的应用（高收益），并且不仅对合成生物学的研究人员，而且对生物工程社区和生物技术行业的研究人员都有巨大的好处。

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardised and interchangeable parts to program cellular behaviour. However, unlike electronic digital circuits, the components in a biological circuit are not connected by wires with physical insulation, and the flow of biological information has to depend on their specific chemical interactions to avoid cross talk. As a result, the same genetic part may not be used twice in one integrated system to prevent the potential unintended interactions between them. Therefore, orthogonal parts and modules are necessary for the compatibility and scalable design of large gene circuits comprising many components. Orthogonality implies that the newly added parts and modules should not cross-talk with those present in the engineered biological systems as well as the host genetic background. Most of the gene circuits constructed so far are small scale systems that have been constructed by costly and inefficient 'trial-and-error' methods with very limited parts. For example, it has taken almost 12 years to progress from the first 3-gene toggle switch to the so far largest constructed 11-gene 4-input AND logic gate in a single cell. A hard truth behind this slowness is that the engineering of complex circuits in living cells is currently limited by the availability of well-characterised and orthogonal (non cross-talk) genetic regulatory building blocks. Hence, an urgent need in synthetic biology is to expand the currently limited toolbox of biological parts with many functional orthogonal elements to scale up our capacity for building large and complex circuits.Nevertheless, it remains a big foundational challenge to expand the range of available orthogonal components in the synthetic biology toolbox. This project aims to address this challenge by developing two novel scalable tools to engineer an expanded library of versatile orthogonal genetic building blocks. In particular, we will build a library of modular and orthogonal genetic NAND and NOR logic gates; these are universal logic gates and their combinations can be used to accomplish any arbitrary complex Boolean logic operations, providing a powerful scalable method for cellular process control. Further, we will create multi-layer genetic programs from different permutations of these engineered logic gates to demonstrate the potential for composing high-order signal processing and transcriptional control functions in a single cell. For example, the engineered genetic programs will be used to implement a high level logic computing device - 1 bit full adder that intake three chemical inputs in specified logic manners to produce two optical outputs. In addition, we will demonstrate that large complex transcriptional control programs can be implemented in a microbial cell factory to precisely and rapidly tune gene expression profiles within the biosynthesis pathway of a high value chemical (violacein).The engineered scalable tools from this study will increase significantly the number of orthogonal control elements, gates and wires in the limited toolbox of synthetic biology, leading to large-scale complex genetic control programs attainable to program advanced behaviours in cells. The successful outcome will lead to a number of applications expected in the biotechnology industry (high gain), and will be of enormous benefit to researchers not only in the synthetic biology and but also in bioengineering communities and those in the biotechnology industry.