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

该项目旨在创造工具，通过使枯草芽孢杆菌依赖于非天然氨基酸来控制特定必需蛋白质的丰度。枯草芽孢杆菌是一种土壤微生物，用于刺激植物生长和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态和分裂的模式系统。协调细胞伸长和分裂的细胞机制包括许多组成部分，其中一些是知之甚少的，并且很少有工具来仔细控制它们的丰度。本项目旨在对枯草芽孢杆菌进行修饰，使目标蛋白的丰度由培养基中提供的非标准氨基酸的浓度控制。这个项目的一个类比是为房间里的灯建造一个调光开关。新的开关允许人们调暗必须保持亮着的灯的亮度，而以前唯一的选择是相对于默认设置增加亮度。如果一个人可以把房间里的灯光调暗，那么他就有可能看到房间里的新特征，并明白保持房间功能所需的光线是多么少。除了这项研究之外，参加特拉华大学新生的国际基因工程机器（iGEM）团队的本科生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项目和促进竞争研究的既定项目（EPSCoR）共同资助。强加的翻译控制允许精确控制表达，包括弱和强的表达比天然启动子在枯草芽孢杆菌。这将补充针对过表达的转录控制的现有方法。本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基酰基tRNA合成酶和tRNA对来比较不同酶系和生物体间琥珀色密码子的抑制。在枯草芽孢杆菌中实现非标准氨基酸掺入后，该项目将研究细胞外氨基酸浓度的滴定能力，并实现模型荧光蛋白的剂量依赖性翻译。该项目旨在利用这种新的控制策略来研究细胞壁的合成，并探索合成营养不良的扩展。细胞形态和长度将被研究，细胞壁合成机制的组成部分被滴定，揭示了实现正常细胞形状所需的蛋白质浓度。合成营养不良是一种很有前途的内在生物控制技术，它使生物体依靠合成营养物质生长。由于这种生物控制技术到目前为止只在大肠杆菌中得到证实，因此该项目将研究是否类似的营养缺陷标记或对细胞壁合成机制的修改可以使生物体对非标准氨基酸的生长产生强大的依赖。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。

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.