Cheng, H. 2018. Using gas-producing enzymes as outputs for synthetic genetic circuits to enable bacterial reporting within complex environmental materials. Dissertation, Rice University, Houston, Texas.
Microbes drive processes in the Earth system far exceeding their physical scale, mediating significant fluxes in biogeochemical cycles. Microbial behavior also affects soil development, water quality, and crop yields. The tools of synthetic biology have the potential to significantly improve our understanding of the roles that microbes play in these processes and the effects of environmental fluctuations on microbial behaviors, which can advance our ability to engineer microbial system for environmental applications, such as bioremediation, waste water treatment, and rhizosphere engineering. However, synthetic biology has not yet been widely used within environmental materials (soils, sediments, and biomass). One of the challenges is that there is a lack of robust and simple-to-detect reporter proteins for nontransparent and heterogeneous materials. Common genetic reporters used to read out circuit status have limited utility for in situ measurements in Earth materials because environmental matrices display high absorbance and auto-fluorescence at the wavelengths of light used for visual reporters like GFP. This technical limitation has made it challenging to use programmed microbes to study how variation in soil environmental parameters (moisture, nutrient status, mineralogy, structure, and temperature) affect real-time biological behaviors.
To overcome this limitation, my thesis research aims to develop a new reporting strategy using gas-producing enzymes, which generate diffusible gases that can be quantified in the headspace of soils using gas chromatography. First, I characterize the activities of two gas-producing enzyme, methyl halide transferase (MHT) and ethylene forming enzyme (EFE), in liquid media and an agricultural soil. Using these two enzymes, gas reporting strains were developed to monitor two dynamic soil microbial processes in situ, horizontal gene transfer and quorum sensing. These proof-of-concept applications demonstrate that the gas-reporting method is a generalizable alternative to study microbial gene expression within soil where visual reporters are not compatible. I envision that this easy-to-use gas reporting method would facilitate the development of more sophisticated synthetic genetic circuits for applications in Earth, environmental, and planetary science.
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