Team:Austin UTexas/Demonstrate

Demonstrate

Click on one of the images below to learn more about our results!

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.

Kombucha Strains

Conjugation

Recapitulation

Ethanol

pH

Kombucha Strains

Successfully isolated microbes from various samples of kombucha.
Identified strains of bacteria and yeast using rRNA gene sequencing.
Characterized each of the isolated microbes to facilitate further experimentation.

Results

Conjugation

Attempted conjugation with G. oxydans.
Performed minimum inhibitory concentration experiments between G. oxydans and spectinomycin, carbenicillin and kanamycin.
Determined that G. oxydans is resistant to spectinomycin and carbenicillin.

Results

Recapitulation

In a process called "recapitulation," we successfully created a kombucha-like culture by adding individual strains of microbes instead of a living culture containing the entire kombucha microbiome.
Determined that the microbe Ga. hansenii is essential for the fermentation of kombucha.
Determined that two distinct strains of the yeast Lachancea fermentati are necessary for the fermentation of kombucha, including one that appears to produce high quantities of C02.

Results

Ethanol

Found literature describing sequences for genes involved in the metabolism of ethanol to acetic acid in the bacterium Ga. hansenii.
Designed Golden Gate parts for the assembly of these genes into a functional construct.
Used a bromothymol blue assay to compare changes in pH resulting from fermentation in multiple strains of Lachancea fermentati isolated from our kombucha.

Results

pH Sensors

Successfully created a neutral pH sensor with a reporter.
Further characterized the P-atp2 Biobrick.
Found literature describing three putative promoters in Gluconobacter oxydans that increase transcription under acidic conditions, and currently characterizing these sequences.

Results

</div>