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

合作研究：使用非标准氨基酸控制枯草芽孢杆菌生长

• 批准号: 2027092
• 负责人: Aditya Kunjapur
• 金额: $ 47.5万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2027092&HistoricalAwards=false
• 关键词: Collaborative; Research; Enabling; control; Bacillus

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.

该项目旨在创建工具，通过使其依赖于非天然氨基酸来控制枯草芽孢杆菌中特定必需蛋白质的丰度。B。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。B。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，其中一些组件知之甚少，并且几乎没有工具来仔细控制它们的丰度。本项目旨在修改B。枯草芽孢杆菌的非标准氨基酸，使得靶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。如果人们可以调暗房间里的光线，那么人们就有可能看到房间里的新功能，并了解保持房间功能所需的光线是多么少。除了这项研究之外，参加特拉华州大学新生的国际遗传工程机器（iGEM）团队的本科生将获得实验室空间和指导，以进行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研究的既定计划（EPSCoR）共同资助。翻译控制的实施允许精确控制表达，包括比B中的天然启动子更弱和更强的表达。枯草杆菌。这将补充现有的方法，是面向转录控制过表达。本项目将探索工程氨酰-tRNA合成酶和tRNA对在大肠杆菌和大肠杆菌中的应用。coli和B.枯草芽孢杆菌中比较跨酶家族和跨生物体的琥珀密码子抑制。在B中实现非标准氨基酸掺入后。该项目将研究滴定胞外氨基酸浓度的能力，并实现模型荧光蛋白的剂量依赖性翻译。该项目的目的是使用这种新的控制策略来询问细胞壁合成，并探索合成营养缺陷型的延伸。细胞形态和长度将作为细胞壁合成机制的组成部分进行研究，从而阐明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有前途的内在生物遏制技术，其中生物体被设计成依赖于合成营养素生长。因为这种生物遏制技术迄今为止只在E.该项目将研究类似的营养缺陷型标记或细胞壁合成机制的修改是否可以实现生物体对其生长的非标准氨基酸的稳健依赖。该奖项反映了NSF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。

项目成果

Incorporation of a Chemically Diverse Set of Non-Standard Amino Acids into a Gram-Positive Organism

将一组化学上多样化的非标准氨基酸掺入革兰氏阳性生物体中

• DOI: 10.21769/bioprotoc.4507
• 发表时间: 2022
• 期刊: BIO-PROTOCOL
• 影响因子: 0.8
• 作者: Stork, Devon;Jones, Michaela;Garner, Ethan C;Kunjapur, Aditya
• 通讯作者: Kunjapur, Aditya

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells

• DOI: 10.1126/sciadv.abf5851
• 发表时间: 2021-06-01
• 期刊: SCIENCE ADVANCES
• 影响因子: 13.6
• 作者: Kunjapur, Aditya M.;Napolitano, Michael G.;Church, George M.
• 通讯作者: Church, George M.