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

通过扩展的工程正交遗传逻辑门和开关库实现合成生物学

• 批准号: BB/N007212/1
• 负责人: Baojun Wang
• 金额: $ 44.13万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN007212%2F1
• 关键词: Enabling; synthetic; biology; expanded; library

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.

合成生物学的一个重要目标是使用标准化和可互换的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的实施。然而，与电子数字电路不同，生物电路中的组件不是通过具有物理绝缘的电线连接的，生物信息的流动必须依赖于它们特定的化学相互作用以避免串扰。因此，同一个基因部分不能在一个集成系统中使用两次，以防止它们之间潜在的非预期相互作用。因此，正交的部件和模块是必要的兼容性和可扩展的设计，包括许多组件的大型基因电路。同源性意味着新添加的部分和模块不应与工程化生物系统中存在的部分和模块以及宿主遗传背景相互干扰。迄今为止，大多数构建的基因电路都是小规模的系统，它们是通过成本高昂且效率低下的“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个基因4输入AND逻辑门已经花费了近12年的时间。这种缓慢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良好表征和正交（非串扰）遗传调控构建模块的可用性的限制。因此，合成生物学迫切需要用许多功能正交元件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，在合成生物学工具箱中扩展可用正交元件的范围仍然是一个很大的基础挑战。该项目旨在通过开发两种新的可扩展工具来解决这一挑战，以设计一个扩展的通用正交遗传构建块库。特别是，我们将建立一个模块化和正交遗传NAND和NOR逻辑门库;这些是通用逻辑门，它们的组合可用于完成任何复杂的布尔逻辑运算，为细胞过程控制提供强大的可扩展方法。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，以展示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位全加器，其以指定的逻辑方式摄取三个化学输入以产生两个光学输出。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现，以精确和快速地调整高价值化学品生物合成途径中的基因表达谱来自本研究的工程化可扩展工具将显著增加合成生物学的有限工具箱中的正交控制元件、门和线的数量，从而导致可实现的大规模复杂遗传控制程序，以编程细胞中的高级行为。成功的结果将导致生物技术行业（高增益）预期的许多应用，不仅对合成生物学研究人员，而且对生物工程界和生物技术行业的研究人员都有巨大的好处。

项目成果

Front Cover: Synthetic Biology Enables Programmable Cell-Based Biosensors (ChemPhysChem 2/2020)

封面：合成生物学使可编程的基于细胞的生物传感器成为可能 (ChemPhysChem 2/2020)

• DOI: 10.1002/cphc.201901192
• 发表时间: 2020
• 期刊: ChemPhysChem
• 影响因子: 2.9
• 作者: Hicks M

A Novel Eukaryote-Like CRISPR Activation Tool in Bacteria: Features and Capabilities

• DOI: 10.1002/bies.201900252
• 发表时间: 2020-04-20
• 期刊: BIOESSAYS
• 影响因子: 4
• 作者: Liu, Yang;Wang, Baojun
• 通讯作者: Wang, Baojun

A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction.

• DOI: 10.1038/s41467-021-22404-9
• 发表时间: 2021-04-13
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Ho TYH;Shao A;Lu Z;Savilahti H;Menolascina F;Wang L;Dalchau N;Wang B
• 通讯作者: Wang B

Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria

• DOI: 10.1038/s41467-019-11479-0
• 发表时间: 2019-08-26
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Liu, Yang;Wan, Xinyi;Wang, Baojun
• 通讯作者: Wang, Baojun