Collaborative Research: Enabling control of Bacillus subtilis growth using non-standard amino acids

该项目旨在创建工具，通过使枯草芽孢杆菌中特定必需蛋白质依赖于非天然氨基酸来控制它们的丰度。枯草芽孢杆菌是一种土壤微生物，用于刺激植物生长并改善动物和人类的肠道健康。枯草芽孢杆菌也是研究细胞形状和分裂的模型系统。协调细胞伸长和分裂的细胞机制包括许多组件，其中一些组件我们知之甚少，并且几乎没有工具可以仔细控制它们的丰度。该项目旨在修改枯草芽孢杆菌，以便通过培养基中提供的非标准氨基酸的浓度来控制目标蛋白的丰度。该项目的一个类比是为房间内的灯建造调光开关。新的开关允许人们调暗必须保持打开状态的灯的亮度，而以前唯一的选择是相对于默认设置增加亮度。如果可以调暗房间内的灯光，那么人们就有可能会看到房间内的新功能，并了解保持房间功能所需的光线有多么少。除了这项研究之外，参与特拉华大学新生国际基因工程机器 (iGEM) 团队的本科生还将获得实验室空间和指导，以开展与非标准氨基酸相关的项目。该项目由系统与合成生物学计划和刺激竞争性研究既定计划 (EPSCoR) 联合资助。翻译控制的实施可以精确控制表达，包括比枯草芽孢杆菌中的天然启动子更弱和更强的表达。这将补充现有的针对过度表达的转录控制的方法。该项目将探索在大肠杆菌和枯草芽孢杆菌中使用工程化的氨酰基-tRNA 合成酶和 tRNA 对来比较跨酶家族和跨生物体的琥珀密码子抑制。在枯草芽孢杆菌中实现非标准氨基酸掺入后，该项目将研究滴定细胞外氨基酸浓度并实现模型荧光蛋白的剂量依赖性翻译的能力。该项目旨在利用这种新的控制策略来询问细胞壁的合成并探索合成营养缺陷型的扩展。随着细胞壁合成机制的组成部分被滴定，细胞形态和长度将被研究，从而揭示实现正常细胞形状所需的蛋白质浓度。合成营养缺陷型是一种很有前途的内在生物遏制技术，其中生物体被设计为依赖合成营养素来生长。由于这种生物遏制技术迄今为止仅在大肠杆菌中得到证实，因此该项目将检查类似的营养缺陷型标记或对细胞壁合成机制的修改是否可以实现生物体对非标准氨基酸生长的强烈依赖。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project seeks to create tools that control the abundance of specific essential proteins in Bacillus subtilis by making them depend on unnatural amino acids. B. subtilis is a soil-dwelling microbe used to stimulate plant growth and to improve intestinal health in animals and humans. B. subtilis is also a model system for studying cell shape and division. The cellular machinery that orchestrates cell elongation and division include numerous components, several of which are poorly understood, and for which there are few tools to carefully control their abundance. This project aims to modify B. subtilis such that the abundance of target proteins is controlled by the concentration of non-standard amino acid supplied in the culture media. An analogy for this project is the construction of a dimmer switch for a light in a room. The new switch allows one to dim the brightness of a light that must stay on, where previously the only option was to increase brightness relative to the default setting. If one can dim the light in a room, then it is possible that one will see new features in the room and understand how little light is needed to keep the room functional. In addition to this research, undergraduate students who participate in the nascent International Genetically Engineered Machines (iGEM) team at the University of Delaware will receive lab space and mentoring to conduct projects related to non-standard amino acids. This project is jointly funded by the Systems and Synthetic Biology program and the Established Program to Stimulate Competitive Research (EPSCoR).The imposition of translational control allows precise control of expression, including weaker and stronger expressions than natural promoters in B. subtilis. This will complement existing approaches that are geared towards transcriptional control for overexpression. This project will explore the use of engineered aminoacyl-tRNA synthetase and tRNA pairs in both E. coli and B. subtilis to compare amber codon suppression across enzyme families and across organism. Upon achievement of non-standard amino acid incorporation in B. subtilis, the project will investigate the ability to titrate extracellular amino acid concentration and achieve dose-dependent translation of a model fluorescent protein. This project then aims to use this new control strategy to interrogate cell wall synthesis and to explore the extension of synthetic auxotrophy. Cell morphology and length will be studied as components of the cell wall synthesis machinery are titrated, shedding light on what protein concentrations are required to achieve normal cell shapes. Synthetic auxotrophy is a promising intrinsic biological containment technique where an organism is engineered to depend on a synthetic nutrient for its growth. Because this biocontainment technique has only been demonstrated thus far in E. coli, this project will examine whether similar auxotrophic markers or the modifications to cell wall synthesis machinery can achieve robust reliance of the organism on non-standard amino acids for its growth.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.