Difference between revisions of "Team:Austin UTexas/Demonstrate"

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

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

1. BIT-China-2015
2. 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.
3. 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.
4. 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
5. 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.
6. 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.