|
|
| Line 93: |
Line 93: |
| | | | |
| | <div id="section4" class = "naviSection"> | | <div id="section4" class = "naviSection"> |
| − | <h2> Ethanol </h2> | + | <h2>Ethanol</h2> |
| − | <P> During the fermentation process, yeast in kombucha produce ethanol, the type of alcohol present in beer, wine, and other alcoholic beverages. This presents a challenge to kombucha brewers who wish to market their product as a non-alcoholic beverage. If the alcohol content of a manufacturer’s kombucha exceeds 0.5% at any point during production, the manufacturer may not market their beverage as non-alcoholic and must be regulated as a producer of alcoholic beverages.<sup>1</sup> One way to tackle this problem with synthetic biology is to ferment with yeast that produce less ethanol. However, this may be impractical. Some bacteria in the SCOBY oxidize ethanol produced by the yeast to produce acetic acid, which is a major component of the beverage’s distinctive, tart flavor. </p>
| + | |
| − | <p>Another approach is to increase the rate at which the bacteria convert the ethanol to acetic acid. Two enzymes are responsible for this process: an alcohol dehydrogenase and an aldehyde dehydrogenase.<sup>2</sup> Using Golden Gate assembly, we plan to assemble a construct containing the coding sequences for these genes and insert the construct into <i>Gluconacetobacter hansenii</i>, an acetic acid-producing bacterium similar to those found in kombucha. Then, we plan to recapitulate kombucha with both the transformed and control <i>Ga. hansenii</i> to evaluate the ethanol content over the course of the fermentation with gas chromatography-mass spectrometry. We also plan to determine whether increasing the acetic acid production will lead to a pH change that could affect the flavor of the beverage by testing the pH and observing the cultures for visible differences. If we are able to create a microbial community that results in a lower ethanol content within the kombucha during fermentation, kombucha brewers could use the modified bacterium to help ensure the ethanol content of their product stays below the legal limit.
| + | |
| − | </p>
| + | |
| | | | |
| − | <h3>References</h3>
| + | </html> |
| − | <ol type="1"> | + | *Found literature describing sequences for genes involved in the metabolism of ethanol to acetic acid in the bacterium <i>Ga. hansenii</i>. |
| − | <li>Alcohol and Tobacco Tax and Trade Bureau. Kombucha Information and Resources. 2016. https://www.ttb.gov/kombucha/kombucha-general.shtml</li>
| + | *Designed Golden Gate parts for the assembly of these genes into a functional construct. |
| − | <li>Mamlouk, D., and Gullo, M. (2013) Acetic Acid Bacteria: Physiology and Carbon Sources Oxidation. <i>Indian Journal of Microbiology</i> 53, 377–384.</li>
| + | *Used a bromothymol blue assay to compare changes in pH resulting from fermentation in multiple strains of <i>Lachancea fermentati</i> isolated from our kombucha. |
| − | </ol> | + | NEED LINK |
| | + | <html> |
| | </div> | | </div> |
| | | | |
| Line 117: |
Line 115: |
| | | | |
| | <div id="section6" class = "naviSection"> | | <div id="section6" class = "naviSection"> |
| − | <h2> pH Sensors </h2> | + | <h2>pH Sensors</h2> |
| − | <p>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.<sup>1,3,5,6</sup> These constructs have been or will be transformed into <i>Escherichia coli</i> to confirm pH sensitivity prior to introduction to kombucha and to see if these constructs could be utilized as sensors in mediums besides kombucha.</p>
| + | |
| − | <p>Modification of <i>Gluconobacter oxydans</i>, 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.<sup>2</sup> Golden Gate assembly is currently being used to quickly assemble these promoters upstream of Venus (pYTK033).<sup>4</sup> Once successful, these pH-sensitive promoters with different reporters will be used to visualize the different members of the kombucha microbiome overtime.</p>
| + | |
| | | | |
| − | <h3>References</h3>
| + | </html> |
| − | <ol type="1">
| + | *Successfully created a neutral pH sensor with a reporter. |
| − | <html><li><a href="https://2015.igem.org/Team:BIT-China/Parts">BIT-China-2015</a></li></html>
| + | *Further characterized the P-atp2 Biobrick. |
| − | <li>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. <i>Journal of Biotechnology 157</i>, 359–372.</li>
| + | *Found literature describing three putative promoters in <i>Gluconobacter oxydans</i> that increase transcription under acidic conditions, and currently characterizing these sequences. |
| − | <li>Kuper, C., and Jung, K. (2005) CadC-mediated activation of the cadBA promoter in Escherichia coli. <i>Journal of Molecular and Microbiological Biotechnology 1</i>, 26–39.</li>
| + | NEED LINK |
| − | <li>Lee ME, DeLoache, WC A, Cervantes B, Dueber, JE. (2015) A Highly-characterized Yeast Toolkit for Modular, Multi-part Assembly. <i>ACS Synthetic Biology 4</i> 975-986</li> | + | <html> |
| − | <li>Nakayama, S.-I., and Watanabe, H. (1998) Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene. <i>Journal of Bacteriology 180</i>, 3522–3528.</li>
| + | </div> |
| − | <li>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. <i>Journal of Bacteriology 177</i>, 5062–5069.</li>
| + | </div> |
| | | | |
| | <!-- | | <!-- |
