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

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<h2> Kombucha Strains </h2>
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<h2>Kombucha Strains</h2>  
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<p> One of the earliest goals of our project was to identify a specific set of microbes responsible for the production of kombucha. To do this, samples of store-bought kombucha were plated onto a variety of media with various dilutions to isolate microbes. Then, morphologically different colonies were cultured and frozen in glycerol for further use. Once we obtained a collection of microbial isolates, each microbe was sequenced and identified using polymerase chain reaction (PCR) to amplify a particular ribosomal RNA gene. The 16S gene was selected for bacterial strains, and the ITS gene was amplified for the fungal samples.<sup>1</sup> After the samples were sequenced, we utilized the <a href="http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp">Ribosomal Database Project (RDP) SeqMatch Tool</a> to identify our isolated species of bacteria and yeast. By identifying these kombucha strains, we were able to use our own experimentally isolated strains for our future kombucha experiments.
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<h3>References</h3>
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*Successfully isolated microbes from various samples of kombucha.
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<li>Marsh, A. J., O'Sullivan, O., Hill, C., Ross, R. P., and Cotter, P. D. (2014) Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples. Food Microbiology.
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*Identified strains of bacteria and yeast using rRNA gene sequencing.
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*Characterized each of the isolated microbes to facilitate further experimentation.  
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section1">Results </a>
 
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<h2> Conjugation </h2>
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<h2>Conjugation</h2>  
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<p> In order to demonstrate that we can genetically engineer the bacterial strains that we identified, <i>Gluconobacter oxydans</i> and <i>Gluconacetobacter xylinus</i>, we attempted to conjugate various plasmids encoding fluorescent devices, such as GFP and Crimson, into these bacteria using a DAP (Diaminopimelic Acid) auxotroph strain of <i>E. coli</i> [REFERENCES].  To assist in this process, we also conducted minimal inhibitory concentration studies with each of these bacteria using [THESE ANTIBIOTICS].  Ultimately, [BLAH BLAH BLAH].  The plasmid, pBTK520, contains GFP and a spectinomycin resistance gene. Once the bacteria are successfully conjugated we can.... [BLAH BLAH BLAH].
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*Attempted conjugation with <i>G. oxydans</i>.
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*Performed minimum inhibitory concentration experiments between <i>G. oxydans</i> and spectinomycin, carbenicillin and kanamycin.
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*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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<h2>Recapitulation </h2>
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<h2>Recapitulation</h2>
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<p>One of the primary focuses of our project is to study the nature of the symbiotic community of fermenting kombucha. Recapitulation refers to the reformation of kombucha by singly adding isolated microbes to a mixture of black tea, sucrose, and water similar to the mixture used in home-brewing practices. Through this process we determined that the microbial community could be recreated from its constituent bacteria and yeast. We also identified the microbes that appear to be vital for the proper recapitulation of kombucha. However, because we cannot taste our lab-brewed kombucha, these conclusions are solely based on qualitative observations. Successful recapitulations indicate that it is in fact possible to produce kombucha with known microbes rather then simply propagating new kombucha from a previous batch. These results elucidate the symbiotic relationships that must exist in order for kombucha to form. Future research may allow us to create kombucha with distinct flavor profiles by varying the combination of strains added to the brew. <p>
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*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.
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*Determined that the microbe <i>Ga. hansenii</i> is essential for the fermentation of kombucha.
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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.
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section3">Results </a>
 
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<h2> Ethanol </h2>
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<h2>Ethanol</h2>
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<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>
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<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.
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<h3>References</h3>
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*Found literature describing sequences for genes involved in the metabolism of ethanol to acetic acid in the bacterium <i>Ga. hansenii</i>.
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<li>Alcohol and Tobacco Tax and Trade Bureau. Kombucha Information and Resources. 2016. https://www.ttb.gov/kombucha/kombucha-general.shtml</li>
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*Designed Golden Gate parts for the assembly of these genes into a functional construct.
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<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>
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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.
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section4">Results </a>
 
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<h2> Brazzein </h2>
 
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<P> One of the potential methods to create designer kombucha is to add a brazzein gene into the bacterial strains. Brazzein, a protein found in the pulp of the edible fruit of the African plant <i>Pentadiplandra brazzeana Baill</i>, is an extremely sweet substance<sup>1</sup>. It is 2,000 times sweeter than sucrose by weight. This makes it a healthy and economical alternative to sugar.  Commercial production of brazzein is limited, however, because it comes from a tropical plant. If it could be more easily harvested, it could be used to improve the flavor of various foods and drinks, including kombucha. By genetically engineering the brazzein gene into the bacteria in kombucha, the drink could be sweetened without adding sugar or excessive calories.  While still being a GMO product, this beverage would be low in sugar and could appeal to a health-conscious consumer.</p>
 
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<h3>References</h3>
 
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<li>Yan, Sen et al. “Expression of Plant Sweet Protein Brazzein in the Milk of Transgenic Mice.” Ed. Xiao-Jiang Li. PLoS ONE 8.10 (2013): e76769. </li>
 
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<li>Brazzein protein structure acquired from European Bioinformatics Institute </li>
 
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Revision as of 04:54, 19 October 2016

Demonstrate

Click the images below to learn more about our results!

Kombucha Strains

Conjugation

Recapitulation

Ethanol

pH