Demonstrate
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Kombucha is a beverage made when a symbiotic community of bacteria and yeast ferments sugared tea. Although kombucha has been consumed for thousands of years in the East, the drink has enjoyed a recent surge in popularity.1 Several kombucha breweries operate in Austin, Texas, our team’s hometown. The role microbes play in the production of the beverage has led our team to wonder if synthetic biology could allow us to create “designer kombucha” with enhanced properties, such as more appealing flavors or additional nutrients. In order to do so, our team attempted to isolate the strains responsible for the fermentation of kombucha, identify them, genetically modify them, and add the individual strains into tea media to recreate the drink. We additionally considered potential applications of the ability to genetically modify the microbial population of kombucha, such as reducing the ethanol content of the beverage and improving taste with brazzein, a sweet-tasting protein. In consideration of Human Practices, we reached out the Austin kombucha community to learn more about what kombucha brewers and consumers would want in a customizable kombucha. Through this interaction, we learned that many kombucha consumers and manufacturers value the traditional, all-natural process of producing the beverage, and that many in the industry would be apprehensive of kombucha made with genetically modified organisms. Though we hope increased public awareness of synthetic biology may someday make a genetically modified kombucha marketable, the current attitudes of kombucha consumers have led us to consider methods of creating designer kombucha that rely only on natural genetic variation.
pH Sensors
Many of the microorganisms involved in the fermentation of kombucha produce acidic metabolites that lower the pH of the culture. Using pH-sensitive promoters to control the expression of reporter proteins, such as GFP or a chromoprotein, would allow visualization of the pH change. The promoters Cpx, P-atp2, and Cadc were selected as pH sensors to indicate pH in the neutral, basic, and acidic ranges, respectively.1,3,5,6 These constructs have been or will be transformed into Escherichia coli to confirm pH sensitivity prior to introduction to kombucha and to see if these constructs could be utilized as sensors in mediums besides kombucha.
Modification of Gluconobacter oxydans, a bacterium in kombucha, is also planned to avoid disturbing the kombucha microbiome. Three endogenous upstream regions of loci that were reported to show increased mRNA synthesis as pH decreased were obtained.2 Golden Gate assembly is currently being used to quickly assemble these promoters upstream of Venus (pYTK033).4 Once successful, these pH-sensitive promoters with different reporters will be used to visualize the different members of the kombucha microbiome overtime.
References
- BIT-China-2015
- Hanke, T., Richhardt, J., Polen, T., Sahm, H., Bringer, S., and Bott, M. (2012) Influence of oxygen limitation, absence of the cytochrome bc1 complex and low pH on global gene expression in Gluconobacter oxydans 621H using DNA microarray technology. Journal of Biotechnology 157, 359–372.
- Kuper, C., and Jung, K. (2005) CadC-mediated activation of the cadBA promoter in Escherichia coli. Journal of Molecular and Microbiological Biotechnology 1, 26–39.
- Lee ME, DeLoache, WC A, Cervantes B, Dueber, JE. (2015) A Highly-characterized Yeast Toolkit for Modular, Multi-part Assembly. ACS Synthetic Biology 4 975-986
- Nakayama, S.-I., and Watanabe, H. (1998) Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene. Journal of Bacteriology 180, 3522–3528.
- Nakayama, S.-I., and Watanabe, H. (1995) Involvement of cpxA, a Sensor of a Two-Component Regulatory System, in the pH-Dependent Regulation of Expression of Shigella sonnei virF Gene. Journal of Bacteriology 177, 5062–5069.
