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

 
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<h2> Demonstrate </h2>
 
<h2> Demonstrate </h2>
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<p>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. </p>
 
<p>Click the images below to learn more about our results!</p>
 
<p>Click the images below to learn more about our results!</p>
 
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<input type="image" src="https://static.igem.org/mediawiki/2016/4/40/T--Austin_UTexas--StrainNavi.png" style="width:100%"; onclick="showOne('section1')"/> <p>Kombucha Strains </p>
 
<input type="image" src="https://static.igem.org/mediawiki/2016/4/40/T--Austin_UTexas--StrainNavi.png" style="width:100%"; onclick="showOne('section1')"/> <p>Kombucha Strains </p>
 
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<input type="image" src="https://static.igem.org/mediawiki/2016/6/64/T--Austin_UTexas--ConjugationPic.png" style="width:100%;" onclick="showOne('section2')" /><p>Conjugation </p>
 
<input type="image" src="https://static.igem.org/mediawiki/2016/6/64/T--Austin_UTexas--ConjugationPic.png" style="width:100%;" onclick="showOne('section2')" /><p>Conjugation </p>
 
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<input type="image" src="https://static.igem.org/mediawiki/2016/0/04/T--Austin_UTexas--RecapNavi.png" style="width:100%;" onclick="showOne('section3')" /><p>Recapitulation</p>
 
<input type="image" src="https://static.igem.org/mediawiki/2016/0/04/T--Austin_UTexas--RecapNavi.png" style="width:100%;" onclick="showOne('section3')" /><p>Recapitulation</p>
 
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<input type="image" src="https://static.igem.org/mediawiki/2016/b/bb/T--Austin_UTexas--EtOHNavi.png" style="width:100%;" onclick="showOne('section4')" /><p>Ethanol</p>
 
<input type="image" src="https://static.igem.org/mediawiki/2016/b/bb/T--Austin_UTexas--EtOHNavi.png" style="width:100%;" onclick="showOne('section4')" /><p>Ethanol</p>
 
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<input type="image" src ="https://static.igem.org/mediawiki/2016/e/e7/T--Austin_UTexas--pHNavi.png" style="width:100%;" onclick="showOne('section6')" /><p>pH</p>
 
<input type="image" src ="https://static.igem.org/mediawiki/2016/e/e7/T--Austin_UTexas--pHNavi.png" style="width:100%;" onclick="showOne('section6')" /><p>pH</p>
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<input type="image" src ="https://static.igem.org/mediawiki/2016/9/92/T--Austin_UTexas--GellanNavi.png" style="width:100%;" onclick="showOne('section7')" /><p>Gellan Gum</p>
 
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section1">Results </a>
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<a href ="https://2016.igem.org/Team:Austin_UTexas/Results#section1">Results</a> (May need to open in a new tab.)
 
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*Determined that <i>G. oxydans</i> is resistant to spectinomycin and carbenicillin.
 
*Determined that <i>G. oxydans</i> is resistant to spectinomycin and carbenicillin.
 
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section2">Results </a>
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section2">Results </a> (May need to open in a new tab.)
 
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*Determined that two distinct strains of the yeast <i>Lachancea fermentati</i> are necessary for the fermentation of kombucha, including one that appears to produce high quantities of C02.
 
*Determined that two distinct strains of the yeast <i>Lachancea fermentati</i> are necessary for the fermentation of kombucha, including one that appears to produce high quantities of C02.
 
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section3">Results </a>
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section3">Results </a> (May need to open in a new tab.)
 
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*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.
 
*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.
 
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section4">Results </a>
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section4">Results </a>( May need to open in a new tab.)
 
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<h2> pH Sensors </h2>
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<h2>pH Sensors</h2>
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<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>
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<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>
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<h3>References</h3>
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*Successfully created a neutral pH sensor with a reporter.
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<html><li><a href="https://2015.igem.org/Team:BIT-China/Parts">BIT-China-2015</a></li></html>
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*Further characterized the P-atp2 Biobrick.
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<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>
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*Found literature describing three putative promoters in <i>Gluconobacter oxydans</i> that increase transcription under acidic conditions, and currently characterizing these sequences.
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<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>
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<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>
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section6">Results</a> (May need to open in a new tab.)
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<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>
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<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>
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<h2>Gellan Gum</h2>
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* Successfully made Gellan Gum plates from <i>Sphingomonas paucimobilis</i>
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* Successfully grew other bacteria on the Gellan Gum plates
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* Shared this DIY technology with the Texas Tech iGEM team
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section7">Results </a> (May need to open in a new tab.)
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Latest revision as of 00:48, 20 October 2016

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

Gellan Gum