Team:Austin UTexas/Demonstrate

Demonstrate

For the gold medal “Demonstrate your work” requirement, we have shown that we can recreate kombucha from scratch by adding isolated strains of microbes to tea media. Our tea media, made with water, black tea, and sucrose, simulates the starting mixture used to brew kombucha. We have performed extensive recapitulation trials to determine which strains of yeast and bacteria are necessary for brewing kombucha. Cataloguing these vital strains is a necessary step toward modifying the starting population to create a “designer beverage” with a variety of properties that could benefit kombucha consumers and manufacturers outside the lab.

Click the images below to learn more about our results!

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 (May need to open in a new tab.)

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 (May need to open in a new tab.)

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 (May need to open in a new tab.)

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 ( May need to open in a new tab.)

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 (May need to open in a new tab.)