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

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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.