Difference between revisions of "Team:H14Z1 Hangzhou/Results"

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{{H14Z1_Hangzhou}}
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    <link rel="stylesheet" type="text/css" href="https://2018.igem.org/Team:H14Z1_Hangzhou/CSS-files/global?action=raw&ctype=text/css" />
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<h1>Results</h1>
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    <link rel="stylesheet" type="text/css" href="https://2018.igem.org/Team:H14Z1_Hangzhou/CSS-files/footer?action=raw&ctype=text/css" />
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<p>Here you can describe the results of your project and your future plans. </p>
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<h3>What should this page contain?</h3>
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<ul>
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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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<h3>Describe what your results mean </h3>
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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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<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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<h3> Project Achievements </h3>
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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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<div class="column third_size" >
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<div class="highlight decoration_A_full">
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<h3>Inspiration</h3>
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<p>See how other teams presented their results.</p>
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<ul>
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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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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </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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</ul>
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</div>
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</div>
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<body>
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    <div id="page_content_container">
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        <div id="menu" attribute="Project"></div>
  
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        <div class="content">
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            <img src="https://static.igem.org/mediawiki/2018/e/e3/T--H14Z1_Hangzhou--head_Results.png" alt="" class="head_div_img" />
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            <div class="content_box">
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                <h1 class="content_title">Results</h1>
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                <div class="content_conts">
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                    <!------------------------part 1------------------------------ -->
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                    <h6 class="content_sub_subtitle">Construction of the expression plasmids</h6>
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                    <p class="content_context">
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                        Three genes ( gshF, metK and cwaA) were cloned to plasmid pNZ8148 to form three modules (GSH
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                        module, SAM module and Adhesion factor module). Each module contained Pnis promoter (containing
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                        RBS sequence), the coding gene and terminator. Then, GSH module and SAM module were combined
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                        together to construct plasmid pNZ-GM. GSH module, SAM module and adhesion factor module were
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                        combined together to construct the final plasmid pNZ-GMcA.
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                    </p>
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                    <p><img style="width: 70%; margin-top: 1em" src="https://static.igem.org/mediawiki/2018/3/3f/T--H14Z1_Hangzhou--project_results_fig1.png"></p>
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                    <p class="content_context" style="text-align:center; font-size:18px">
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                        Figure. 1 Schematic diagram of construction of mulita-functional plasmids.
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                    </p>
  
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                    <!------------------------part 2------------------------------ -->
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                    <h6 class="content_sub_subtitle">Design and integration of three different modules (gshF, met K
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                        and cwaA)</h6>
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                    <p class="content_context">
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                        Two fragments were chosen in the constructed plasmid to validate the validity. Fragment 1
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                        named gm containing gene gshF and metK was 3670 bp and fragment 2 named cA containing gene
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                        cwaA was 3079 bp. As shown in Figure. 2, all the picked colonies contains gene gshF, metK
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                        and cwaA, illustrating that the plasmid pNZ-GMcA was successfully constructed.
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                    </p>
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                    <p><img style="width: 60%; margin-top: 1em; margin-left: 10em" src="https://static.igem.org/mediawiki/2018/f/f2/T--H14Z1_Hangzhou--project_results_fig2.png"></p>
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                    <p class="content_context" style="text-align:center; font-size:18px">
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                        Figure. 2 Validation of plasmid pNZ-GMcA. M represented marker. gm-1 to gm-5 and cA-1 to cA-5
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                        all represented the same five randomly picked colonies.
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                    </p>
  
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                    <!------------------------part 3------------------------------ -->
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                    <h6 class="content_sub_subtitle">SDS-PAGE detection of protein GshF and MetK</h6>
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                    <p class="content_context">
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                        In order to validate the availability of the modules expressed in the plasmid pNZ-GMcA. We
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                        carried out SDS-PAGE analysis of protein GshF and MetK. GshF protein is about 86kDa and MetK
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                        protein is about 43 kDa. As shown in Figure. 3, expected bands of GshF and MetK protein were
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                        observed on the gel. Recombinant <i>L.lactis</i> containing pNZ-GMcA induced with different nisin
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                        concentration showed higher expression of MetK protein than wild-type. These results
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                        illustrated the successfully expression of GSH module and SAM module in plasmid pNZ-GMcA.
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                    </p>
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                    <p><img style="width: 70%; margin-top: 1em" src="https://static.igem.org/mediawiki/2018/2/2b/T--H14Z1_Hangzhou--project_results_fig3.png"></p>
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                    <p class="content_context" style="text-align:center; font-size:18px">
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                        Figure. 3 SDS PAGE validation of gene gshF and metK expression in <i>L.lactis</i>. M represented
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                        marker. WT represented <i>L.lactis</i> NZ9000. 1-3 represented <i>L.lactis</i>/pNZ-GMcA induced with 100,
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                        50 and 20 ng/ml nisin.
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Latest revision as of 01:11, 18 October 2018

<!DOCTYPE html>

Results

Construction of the expression plasmids

Three genes ( gshF, metK and cwaA) were cloned to plasmid pNZ8148 to form three modules (GSH module, SAM module and Adhesion factor module). Each module contained Pnis promoter (containing RBS sequence), the coding gene and terminator. Then, GSH module and SAM module were combined together to construct plasmid pNZ-GM. GSH module, SAM module and adhesion factor module were combined together to construct the final plasmid pNZ-GMcA.

Figure. 1 Schematic diagram of construction of mulita-functional plasmids.

Design and integration of three different modules (gshF, met K and cwaA)

Two fragments were chosen in the constructed plasmid to validate the validity. Fragment 1 named gm containing gene gshF and metK was 3670 bp and fragment 2 named cA containing gene cwaA was 3079 bp. As shown in Figure. 2, all the picked colonies contains gene gshF, metK and cwaA, illustrating that the plasmid pNZ-GMcA was successfully constructed.

Figure. 2 Validation of plasmid pNZ-GMcA. M represented marker. gm-1 to gm-5 and cA-1 to cA-5 all represented the same five randomly picked colonies.

SDS-PAGE detection of protein GshF and MetK

In order to validate the availability of the modules expressed in the plasmid pNZ-GMcA. We carried out SDS-PAGE analysis of protein GshF and MetK. GshF protein is about 86kDa and MetK protein is about 43 kDa. As shown in Figure. 3, expected bands of GshF and MetK protein were observed on the gel. Recombinant L.lactis containing pNZ-GMcA induced with different nisin concentration showed higher expression of MetK protein than wild-type. These results illustrated the successfully expression of GSH module and SAM module in plasmid pNZ-GMcA.

Figure. 3 SDS PAGE validation of gene gshF and metK expression in L.lactis. M represented marker. WT represented L.lactis NZ9000. 1-3 represented L.lactis/pNZ-GMcA induced with 100, 50 and 20 ng/ml nisin.