Difference between revisions of "Team:SZU-China/Results"

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{{SZU-China}}
 
 
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    <title>Procedure</title>
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<h1>Results</h1>
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<p>Here you can describe the results of your project and your future plans. </p>
 
  
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<h5>What should this page contain?</h5>
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<ul>
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    </style>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project. </li>
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<li> Considerations for replicating the experiments. </li>
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</ul>
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<h5>You should also describe what your results mean: </h5>
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    <meta http-equiv="Content-Type" content="text/html; charset=utf-8">
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    <link rel="stylesheet" type="text/css" href="https://2017.igem.org/Template:SZU-China/sub/CSS?action=raw&amp;ctype=text/css" />
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    <link href="https://2017.igem.org/Template:SZU-China/HP/CSS?action=raw&amp;ctype=text/css" rel="stylesheet" type="text/css">
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<ul>
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    <link href='http://fonts.font.im/css?family=Open+Sans' rel='stylesheet' type='text/css'>
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
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    <link href="table.css" type="text/css" rel="stylesheet" />
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
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</ul>
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
 
  
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<ul>
 
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<li>A list of linked bullet points of the successful results during your project</li>
 
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
 
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</ul>
 
  
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<body>
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    <div class="navbar" style="top:5px;position:fixed;z-index:5">
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            <ul id="nav-list-ul" class="nav-list">
  
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                    <a href="#">TEAM</a>
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                    <span class="caret"></span>
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                    <ul class="sub-nav" style="top:-5px">
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Team">MEMBERS</a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Collaborations">COLLABORATIONS</a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Attributions">ATTRIBUTION</a></li>
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                    </ul>
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                </li>
  
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<h5>Inspiration</h5>
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                <li>
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<p>See how other teams presented their results.</p>
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                    <a href="#">PRACTICE</a>
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<ul>
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                    <span class="caret"></span>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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                    <ul class="sub-nav">
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/HP/Gold_Integrated">INTEGRATED HP</a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/HP/Silver">SILVER HP</a></li>
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</ul>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Engagement">ENGAGEMENT</a></li>
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                    </ul>
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                </li>
  
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</div>
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                <li>
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                    <a href="#">EXPERIMENT</a>
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                    <span class="caret"></span>
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                    <ul class="sub-nav">
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Procedure">PROCEDURE</a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Results">RESULTS</a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Demonstrate">DEMONSTRATE</a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Protocol">PROTOCOL</a></li>
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                    </ul>
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                </li>
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                <li>
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                    <a href="#">PROJECT</a>
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                    <span class="caret"></span>
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                    <ul class="sub-nav">
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Description">DESCRIPTION</a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Design">DESIGN</a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Model">MODEL</a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Safety">SAFTY</a></li>
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                    </ul>
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                </li>
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 +
 
 +
                <li>
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                    <a href="#">ACHIEVEMENT</a>
 +
                    <span class="caret"></span>
 +
                    <ul class="sub-nav">
 +
                        <li><a href="https://2017.igem.org/Team:SZU-China/Achievement">MEDAL</a></li>
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                        <li><a href="https://2017.igem.org/Team:SZU-China/Parts">PARTS</a></li>
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                    </ul>
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                </li>
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            </ul>
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    <div id="page" style="position:relative;top:100px;width:100%">
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        <section id="overview" style="padding:96px 0;background-color:white;">
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            <div class="container">
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                        <h3 class="uppercase color-primary mb40 " style="margin-bottom: 40px;font-size:50px"><center>Results</center> </h3>
 +
                        <p class="lead mb40" style="width:60%;margin:0 auto">After transferring the device plasmid, we finally equipped WB800 bacteria with 3 functional gene modules, which have functions of secreting Carbonic anhydrase, accelerating germination and surviving in alkaline environment respectively. We also conducted experiment based on these 3 aspects.</p>
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                    </div>
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                </div>
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            </div>
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        </section>
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        <section class="cd-section" style="padding:96px 0;background-color:rgba(245,245,245,0.45)">
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            <div class="container">
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                <div class="row">
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                    <div class="col-sm-12">
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                        <h3 class="uppercase color-primary mb40 mb-xs-24" style="margin-bottom: 40px;">
 +
                            <center>Carbonic anhydrase</center>
 +
                        </h3>
 +
 
 +
                        <div class="row text-center" style="width:85%;margin:0 auto;text-align:justify">
 +
                           
 +
                            <p class="lead" style="color:black;">We verified the translation of Carbonic anhydrase(CA) by SDS-PAGE protein electrophoresis and Coomassie blue staining. As you can see, the band near 35kDa was α-CA protein which only exist in the transformed bacteria but not in the WB800. We can conclude that the protein was translated successfully.</p><br/>
 +
                           
 +
                            <div style="text-align:center;">
 +
                                <img src="https://static.igem.org/mediawiki/2017/e/e2/T--SZU-China--pe.jpg" style="height:60%"/>
 +
                            </div>
 +
 
 +
                            <center style="padding-top:5px;color:#2f2f2f;font-size:14px">Fig 2. Western blot result of Carbonic anhydrase expression</center>
 +
                           
 +
 
 +
                            <br/><br/>
 +
                          <p class="lead" style="color:black">We determined CA activity according a method modified from Brownell et al. (1991). The CA activity was measured by the different rates of decrease in pH in the system involving 5ml CO2-saturated water, 10ml buffer and 500ul CA samples or Dl water. We calculated the activity according to the formula U= (T0 -Ts )/ T0 ,where T0and Ts represent time for pH change with blank and samples, respectively. By testing different kinds of CA each for several times and taking the average ,we got the CA activity per milliliter of bacteria solution.</p>
 +
 
 +
                           
 +
                            <div style="text-align:center;width:50%;float:left;">
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                                <img src="https://static.igem.org/mediawiki/2017/3/35/T--SZU-China--ca1.jpg" style="height:45%" />
 +
                                <center style="padding-top:5px;color:#2f2f2f;font-size:14px">Fig 2. Western blot result of Carbonic anhydrase expression</center>
 +
                            </div>
 +
                         
 +
                            <div style="text-align: center; width: 50%; float: left;">
 +
                                <img src="https://static.igem.org/mediawiki/2017/2/2b/T--SZU-China--ca2.jpg" style="height:45%" />
 +
                                <center style="padding-top:5px;color:#2f2f2f;font-size:14px">Fig 3. Western blot result of Carbonic anhydrase expression</center>
 +
                            </div>
 +
                            <p style="clear:both"></p>
 +
                            <p></p><br/><br/>
 +
 
 +
                             
 +
                            <p class="lead" style="color:black">Since the fact that CA can also catalyze the hydration reaction of ester,which can be used to assay the activity of CA, we conducted another measurement on CA according to a method from Verpoorte JA(1967).</p><br/>
 +
                            <div style="text-align:center;width:50%;float:left;">
 +
                                <img src="https://static.igem.org/mediawiki/2017/8/86/T--SZU-China--ca3.jpg" style="height:45%" />
 +
                                <center style="padding-top:5px;color:#2f2f2f;font-size:14px">Fig 4. Western blot result of Carbonic anhydrase expression</center>
 +
                            </div>
 +
 
 +
                            <div style="text-align: center; width: 50%; float: left;">
 +
                                <img src="https://static.igem.org/mediawiki/2017/8/8e/T--SZU-China--ca4.jpg" style="height:45%" />
 +
                                <center style="padding-top:5px;color:#2f2f2f;font-size:14px">Fig 5. Western blot result of Carbonic anhydrase expression</center>
 +
                            </div>
 +
                            <p style="clear:both"></p><br/>
 +
                            <p class="lead" style="color:black">From the above results, we may draw the conclusion that transferred strains range a higher level in CA activity.</p>
 +
 
 +
                        </div>
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                        <br /><br /><br />
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                    </div> 
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                <div class="row">
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                    <div class="col-sm-12">
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                        <h3 class="uppercase color-primary mb40 mb-xs-24" style="margin-bottom: 40px;">
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                            <center>Germination</center>
 +
                        </h3>
 +
 
 +
                        <div class="row text-center" style="width:85%;margin:0 auto;text-align:justify">
 +
 
 +
                            <p class="lead" style="color:black;">To verify the gene gerAa for promoting spore germination, we divide the germination detection into two groups: control group (B. subtilis WB800) and gerAa group (B.subtilis WB800 that has gerAa gene with promotor pssPb). Theoretically, through the Germination detection, gerAa group should exhibit higher germination proportion and germination rate than control group, as gerAa is overexpressed. As shown in Fig.1, the germination rate and proportion varied between control group and gerAa group. gerAa group shows a high proportion in germination and rapid rate in germination. Therefore, gene gerAa is verified to be in good condition and can work efficiently.</p><br />
 +
 
 +
                            <div style="text-align:center;">
 +
                                <img src="https://static.igem.org/mediawiki/2017/8/80/T--SZU-China--gerA.jpg" style="height:60%" />
 +
                            </div>
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 +
                            <center style="padding-top:5px;color:#2f2f2f;font-size:14px">Fig 6. The germination experiment of control group and experiment group (induced by 2mmol/L L-alanine)</center>
 +
 
 +
 
 +
                            <br /><br />
 +
                            <p class="lead" style="color:black">We determined CA activity according a method modified from Brownell et al. (1991). The CA activity was measured by the different rates of decrease in pH in the system involving 5ml CO2-saturated water, 10ml buffer and 500ul CA samples or Dl water. We calculated the activity according to the formula U= (T0 -Ts )/ T0 ,where T0and Ts represent time for pH change with blank and samples, respectively. By testing different kinds of CA each for several times and taking the average ,we got the CA activity per milliliter of bacteria solution.</p>
 +
                   
 +
                        </div>
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                        <br /><br /><br />
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 +
                <div class="row">
 +
                    <div class="col-sm-12">
 +
                        <h3 class="uppercase color-primary mb40 mb-xs-24" style="margin-bottom: 40px;">
 +
                            <center>Alkali Resistance</center>
 +
                        </h3>
 +
 
 +
                        <div class="row text-center" style="width:85%;margin:0 auto;text-align:justify">
 +
                           
 +
                            <p class="lead" style="color:black;margin-bottom:5px">To verify the function of TupA for alkaline resistance in our modified baceria, we test the WB800 strain by the following steps:</p><br/>
 +
                            <p class="lead" style="color:black;">At first, we conduct a gene transformation experiment of origin strain WB800, by transforming an extraordinary gene TupA. Then we collect the mono bacteria from the petri dish to culture. We also have a series of assays following up: extracting the plasmids, digesting the plasmids with endonuclease and running through DNA electrophoresis just to verify we have successfully transformed gene TupA into the B.subtilis WB800 (as shown in Fig.7).</p>
 +
 
 +
                            <div style="text-align:center;">
 +
                                <img src="https://static.igem.org/mediawiki/2017/b/bf/T--SZU-China--tupA.jpg" style="height:60%" />
 +
                            </div>
 +
 
 +
                            <center style="padding-top:5px;color:#2f2f2f;font-size:14px">Fig 7. Western blot result of Carbonic anhydrase expression</center>
 +
                           
 +
 
 +
                            <br/><br/>
 +
                          <p class="lead" style="color:black">Further more, we want to know if the B.subtilis WB800 that been transformed can express gene TupA. So we have designed the way to verify:</p>
 +
                            <p class="lead" style="color:#2f2f2f;">1. We divide the bacteria into two groups: one is the control group (B.subtilis WB800), and another is the experiment group (B.subtilis WB800 that includes gene TupA). And culture these two groups with liquid medium.</p>
 +
                            <p class="lead" style="color: #2f2f2f;  ">2. Prepare four types of solid medium with the pH of 9,10 and 11 respectively .</p>
 +
                            <p class="lead" style="color: #2f2f2f; ">3. When the concentration of the B.subtilis from two groups are nearly equal, take 5μL of sample and drop it on the plate(each plate has already been divided into 4 even zones) at the correct zone.(Also we have take 10μL of the sample to repeat). Culture the B.subtilis on the solid plate.</p>
 +
                            <p class="lead" style="color: #2f2f2f; ">4. After 48hours, observe and verify the plate by dyeing with crystal violet before wash the plate with ddH₂O.</p>
 +
                           
 +
                            <div style="text-align:center;width:33.3%;float:left;">
 +
                                <img src="https://static.igem.org/mediawiki/2017/c/c9/T--SZU-China--N9.jpg" style="height:30%" />
 +
                                <p style="padding: 5px 1px 2px 1px; color: #2f2f2f; font-size: 14px">Fig 8. Western blot result of Carbonic anhydrase expression</p>
 +
                            </div>
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 +
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                                <p style="padding: 5px 1px 2px 1px; color: #2f2f2f; font-size: 14px">Fig 9. Western blot result of Carbonic anhydrase expression</p>
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                                <p style="padding:5px 1px 2px 1px;color:#2f2f2f;font-size:14px;">Fig 10. Western blot result of Carbonic anhydrase expression</p>
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                            <p class="lead" style="color:black">So we have come to the conclusion: gene TupA can regulate the alkaline resistance of B.subtilis.</p>
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Revision as of 11:48, 30 October 2017

Procedure

Results

After transferring the device plasmid, we finally equipped WB800 bacteria with 3 functional gene modules, which have functions of secreting Carbonic anhydrase, accelerating germination and surviving in alkaline environment respectively. We also conducted experiment based on these 3 aspects.

Carbonic anhydrase

We verified the translation of Carbonic anhydrase(CA) by SDS-PAGE protein electrophoresis and Coomassie blue staining. As you can see, the band near 35kDa was α-CA protein which only exist in the transformed bacteria but not in the WB800. We can conclude that the protein was translated successfully.


Fig 2. Western blot result of Carbonic anhydrase expression


We determined CA activity according a method modified from Brownell et al. (1991). The CA activity was measured by the different rates of decrease in pH in the system involving 5ml CO2-saturated water, 10ml buffer and 500ul CA samples or Dl water. We calculated the activity according to the formula U= (T0 -Ts )/ T0 ,where T0and Ts represent time for pH change with blank and samples, respectively. By testing different kinds of CA each for several times and taking the average ,we got the CA activity per milliliter of bacteria solution.

Fig 2. Western blot result of Carbonic anhydrase expression
Fig 3. Western blot result of Carbonic anhydrase expression



Since the fact that CA can also catalyze the hydration reaction of ester,which can be used to assay the activity of CA, we conducted another measurement on CA according to a method from Verpoorte JA(1967).


Fig 4. Western blot result of Carbonic anhydrase expression
Fig 5. Western blot result of Carbonic anhydrase expression


From the above results, we may draw the conclusion that transferred strains range a higher level in CA activity.




Germination

To verify the gene gerAa for promoting spore germination, we divide the germination detection into two groups: control group (B. subtilis WB800) and gerAa group (B.subtilis WB800 that has gerAa gene with promotor pssPb). Theoretically, through the Germination detection, gerAa group should exhibit higher germination proportion and germination rate than control group, as gerAa is overexpressed. As shown in Fig.1, the germination rate and proportion varied between control group and gerAa group. gerAa group shows a high proportion in germination and rapid rate in germination. Therefore, gene gerAa is verified to be in good condition and can work efficiently.


Fig 6. The germination experiment of control group and experiment group (induced by 2mmol/L L-alanine)


We determined CA activity according a method modified from Brownell et al. (1991). The CA activity was measured by the different rates of decrease in pH in the system involving 5ml CO2-saturated water, 10ml buffer and 500ul CA samples or Dl water. We calculated the activity according to the formula U= (T0 -Ts )/ T0 ,where T0and Ts represent time for pH change with blank and samples, respectively. By testing different kinds of CA each for several times and taking the average ,we got the CA activity per milliliter of bacteria solution.




Alkali Resistance

To verify the function of TupA for alkaline resistance in our modified baceria, we test the WB800 strain by the following steps:


At first, we conduct a gene transformation experiment of origin strain WB800, by transforming an extraordinary gene TupA. Then we collect the mono bacteria from the petri dish to culture. We also have a series of assays following up: extracting the plasmids, digesting the plasmids with endonuclease and running through DNA electrophoresis just to verify we have successfully transformed gene TupA into the B.subtilis WB800 (as shown in Fig.7).

Fig 7. Western blot result of Carbonic anhydrase expression


Further more, we want to know if the B.subtilis WB800 that been transformed can express gene TupA. So we have designed the way to verify:

1. We divide the bacteria into two groups: one is the control group (B.subtilis WB800), and another is the experiment group (B.subtilis WB800 that includes gene TupA). And culture these two groups with liquid medium.

2. Prepare four types of solid medium with the pH of 9,10 and 11 respectively .

3. When the concentration of the B.subtilis from two groups are nearly equal, take 5μL of sample and drop it on the plate(each plate has already been divided into 4 even zones) at the correct zone.(Also we have take 10μL of the sample to repeat). Culture the B.subtilis on the solid plate.

4. After 48hours, observe and verify the plate by dyeing with crystal violet before wash the plate with ddH₂O.

Fig 8. Western blot result of Carbonic anhydrase expression

Fig 9. Western blot result of Carbonic anhydrase expression

Fig 10. Western blot result of Carbonic anhydrase expression



So we have come to the conclusion: gene TupA can regulate the alkaline resistance of B.subtilis.




Conclusion

Based on upstream experiment on genetic engineering level, our genetic modified WB800 successfully achieved the 3 functions needed for concrete self-repairing sysyem. Next, we will measure their true performance when embedded in the concrete as form of micro-capsule. See the DEMONSTRATE page for our downstream results.





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