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

 
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{{Austin_UTexas}}
 
{{Austin_UTexas}}
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.content {
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|+Microbes Isolated and Identified from Various Store Bought Kombucha Samples
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! |Species
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! |Classification
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! |Brand of Kombucha Isolated From
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|-style="text-align: center;"
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|Staphylococcus warneri
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|Bacteria
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|GT’s Kombucha
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|-style="text-align: center;"
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|Staphylococcus epidermidis
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|Bacteria
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|GT's Kombucha
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|-Yeast
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|-style="text-align: center;"
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|Gluconobacter oxydans*
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|Bacteria
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|GT’s Kombucha
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|-style="text-align: center;"
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|Lachancea fermentati*
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|Yeast
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|Buddha's Brew
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|-style="text-align: center;"
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|Propionibacterium acnes
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|Bacteria
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|Buddha's Brew
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|-style="text-align: center;"
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|Micrococcus luteus
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|Bacteria
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|Buddha's Brew
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|-style="text-align: center;"
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|Bacillus pumilus
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|Bacteria
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|Buddha's Brew
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|-style="text-align: center;"
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|Saccharomyces cerevisiae
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|Yeast
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|LIVE Soda Kombucha
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|-style="text-align: center;"
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|Schizosaccharomyces pombe*
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|Yeast
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|LIVE Soda Kombucha
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(*Indicates a species that is considered vital to the production of kombucha)
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<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>
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<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>
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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>
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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>
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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>
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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>
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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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<br>
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<div id="section1" class= "naviSection">
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<h2>Kombucha Strains</h2>
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*Successfully isolated microbes from various samples of kombucha.
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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/File:T--Austin_UTexas--RecapitulationsDay1vDay4.jpg" class="image">
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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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<img alt="T--Austin UTexas--RecapitulationsDay1vDay4.jpg" src="https://static.igem.org/mediawiki/2016/4/40/T--Austin_UTexas--RecapitulationsDay1vDay4.jpg" width="80%">
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<h2>Conjugation</h2>
  
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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> (May need to open in a new tab.)
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<h2>Recapitulation</h2>
  
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</html>
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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> (May need to open in a new tab.)
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<div id="section4"  class = "naviSection">
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<h2>Ethanol</h2>
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</html>
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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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*Designed Golden Gate parts for the assembly of these genes into a functional construct.
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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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<html>
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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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</div>
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<div id="section6"  class = "naviSection">
 
<h2>pH Sensors</h2>
 
<h2>pH Sensors</h2>
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Possessing the ability to monitor the brewing process of kombucha without disturbing the microenvironment and using a very visible color reporter would allow for greater insights as to how the populations of organisms and pH may change due to competition amongst other bacteria and yeast in the beverage and SCOBY of the kombucha. The byproducts produced by the kombucha as it brews causes the tea to become more acidic, leading to our team searching for pH sensitive promoters, and for ways to implement these into kombucha.
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*Successfully created a neutral pH sensor with a reporter.
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<p>
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*Further characterized the P-atp2 Biobrick.
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Though an acidic sensor was what was required for our kombucha analysis, the identification of sensors in other areas of the pH spectrum were explored as well. Three sequences were identified, the CadC operon for the acidic range, CpxA-CpxR complex for the neutral range, and the P-atp2 promoter from the BioBrick Registry (<a href="http://parts.igem.org/Part:BBa_K1675021">BBa_K1675021</a>) for the basic range. Each sequence was paired with a unique corresponding reporter sequence so that if each pH sensitive plasmid were in the same environment, the specific pH of the system could be seen. The reporters used were, <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> for the CadC construct, <a href="http://parts.igem.org/Part:BBa_K1033916">BBa_K1033916</a> for the CpxA-CpxR complex, and <a href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a> for the P-atp2 promoter.
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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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<u>CadC</u>
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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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<p>
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The CadC operon is a native pathway in <i>E. coli</i>, involved in the cadaverine synthesis pathway. The protein CadC protein on the operon is produced and activates segments downstream of the operon on the CadBA receptors. The CadC protein is pH sensitive to an external pH 5.5 and below, as well as lysine dependent. A point mutation on codon 265, in which argenine is converted to cystine, causes the CadC protein to become lysine independent (Dell, Neely, Olson, 1994).
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<p>
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Unfortunately, we have been unable to grow the modified CadC operon in <i>E. coli</i> suggesting some form of cell toxicity. Due to this apparent toxicity, no data regarding this mutant CadC could be collected. Alternative candidates are being explored for other pH sensors that sense in the acidic range.
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<u>CpxA-CpxR</u>
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<p>
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CpxA-CpxR is a two-component mechanism that is activated at pH 7.4 and repressed at pH 6.0. CpxA is an intermembrane protein that autophosphorylates at a certain external pH, CpxR (a kinase) then gets phosphorylated by CpxA and acts as a transcription factor. This system originally is a transcription factor for the virF gene, but we replaced virF with the Reporter. The original sequence was found in Shigella sonnei, but E. coli has a homolog of these proteins so all that is required on the construct is the appropriate prefix/suffix and CpxR binding site.
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<a href=https://static.igem.org/mediawiki/parts/0/05/T--Austin_UTexas--Cpx_pH_Culture_Tubes.png" class="image>
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<p>
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The order from left to right is Control pH 6-9 and then Experimental pH 6-9. These are the show the change in expression accordingly with the change of pH. The main point is that the Control at pH 6 has more expression of the Yellow-Green Chromoprotein than the Experimental at pH 6. The pH-Dependent promoter of the Experimental group is down-regulated at pH 6 whereas the control is not. The normalized data is found in the Parts page.
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<u>P-atp2</u>
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<p>
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The P-atp2 promoter, native to the bacterium <i>Corynebacterium glutamicum</i> is reportedly induced at pH 7, to pH 9 (XX_how to site another iGEM team?_XX). Utilizing the blue chromoprotein (<a href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>), a test was designed in which a plasmid containing the P-atp2 promoter with the blue chromoprotein was grown alongside an <i>E. coli</i> line that contained a plasmid with just the blue chromoprotein. We expected to see constant blue chromoprotein production in the control series (those that lacked P-atp2) and a visual increase in blue chromoprotein as the pH was raised from 6 to 9 in the cells that contained the P-atp2 construct.
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<img alt="T--Austin UTexas--Patp2Results.jpg" src="https://static.igem.org/mediawiki/2016/4/46/T--Austin_UTexas--Patp2Results.png" width="80%">
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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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</div>
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{{Team:Austin_UTexas/Footer}}
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<!-- Commented out to prevent from being visible on page
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<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
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<h5>What should this page contain?</h5>
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<ul>
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<li> A clear and concise description of your project.</li>
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<li>A detailed explanation of why your team chose to work on this particular project.</li>
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<li>References and sources to document your research.</li>
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<li>Use illustrations and other visual resources to explain your project.</li>
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</ul>
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<h5>Advice on writing your Project Description</h5>
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<p>
 
<p>
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<u>Next Steps and the GOX Sequences as Putative Promoters</u>
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We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be consist, accurate and unambiguous in your achievements.
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</p>
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<h2>Ethanol Reduction</h2>
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<p>
 
<p>
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<u>Identifying genes of interest</u>
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Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
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<p>
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</p>
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In order to design a construct increasing expression of PQQ-ADH and ALDH in <i>Ga. hansenii</i>, it was necessary to find the genome of the ATCC strain and identify the coding sequences for these genes. The whole genome shotgun sequence for our organism, ATCC 53582, is published on NCBI by J. Abbot (2015) with annotations regarding the functions of specific sequences. Coding sequences are annotated with proposed gene products. Though there are several aldehyde dehydrogenase genes annotated in the genome, there is only one which is described as membrane-bound, matching the description from Mamlouk and Gullo (2013). There are additionally multiple alcohol dehydrogenases. A known amino acid sequence for a homologous PQQ-ADH in Comamonas testosteroni was compared against sequences in the Ga. hansenii genome using BLAST (Table 1,). One ADH enzyme found in the <i>Ga. hansenii</i> genome sequence matches the <i>C. testosteroni</i> sequence with a query cover value of 94% and an E value of 0 (third line of table 1).
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<p>*Need to insert table 1 here*
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<p>
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<u>Creation of Golden Gate parts</u>
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<p>
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In order to assemble the construct, the coding sequences for the genes of interest must be amplified from the <i>Ga. hansenii</i> genome and edited such that they have the correct Golden Gate overhangs and no internal BsaI or BsmBI restriction sites. The sequences were uploaded to Benchling for analysis and planning. The coding sequence for the membrane-bound ALDH contains a BsaI restriction site near the middle of the gene (Figure 1), and the PQQ-ADH coding sequence contains a BsmBI restriction site near the end of the gene (Figure 2). To eliminate the BsaI site in ALDH, primers were designed that would introduce a point mutation at the restriction site. One set of primers, igem2016_KOM_EtOH_01 and igem2016_KOM_EtOH_02, amplifies the sequence upstream of the restriction site, adding a type 3 Golden Gate prefix and removing the restriction site. Another set, igem2016_KOM_EtOH_03 and igem2016_KOM_EtOH_04, amplifies the region downstream of the restriction site, introducing a mutation to the site and adding a type 3 Golden Gate suffix to the end of the gene. These two products will be used in an overlap PCR reaction to create a final product with no BsaI restriction sites and the correct prefix and suffix for assembly. To remove the BsmBI site from the PQQ-ADH coding sequence, a set of primers (igem2016_KOM_EtOH_05 and igem2016_KOM_EtOH_06) was designed to amplify the region upstream of the restriction site and add a Golden Gate type 3 prefix to the beginning of the sequence. The reverse primer additionally adds a mutation to existing BsmBI restriction site and creates a new BsmBI restriction site that will be used to join the piece to a double-stranded DNA, igem2016_KOM_EtOH_07, containing the rest of the gene’s coding sequence appended with a Golden Gate type 3 suffix. The assembly of the PQQ-ADH part will therefore take place in two reactions: one reaction in which the upstream piece of DNA is created, and one reaction in which it is ligated to the gBlock. Table 2 contains more information about each of these oligonucleotides. All were ordered from IDT.
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<p> *Will need to insert additional tables and figures. Numbering of tables and figures may need to be adjusted. If anyone is willing to help insert figures and tables in this section, contact me (Stratton) and I'll send everything to be inserted.
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<h5>References</h5>
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<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you thought about your project and what works inspired you.</p>
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</div>
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<h5>Inspiration</h5>
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<p>See how other teams have described and presented their projects: </p>
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<ul>
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<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
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<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
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<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
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</ul>
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</div>
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  <h2>Demonstrate</h2>
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    <p> Click on one of 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>
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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>
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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>
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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>
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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>
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<h2>Kombucha Strains</h2>
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*Successfully isolated microbes from various samples of kombucha.
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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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<html>
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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section1">Results </a>
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<div id="section2" class = "naviSection">
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<h2>Conjugation</h2>
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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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*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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*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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*Designed Golden Gate parts for the assembly of these genes into a functional construct.
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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>pH Sensors</h2>
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*Successfully created a neutral pH sensor with a reporter.
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*Further characterized the P-atp2 Biobrick.
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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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<a href = "https://2016.igem.org/Team:Austin_UTexas/Results#section6">Results </a></div>
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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