View source for Team:H14Z1 Hangzhou/Results

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                <h1 class="content_title">Results</h1>
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                    <!------------------------part 1------------------------------ -->
                    <h6 class="content_sub_subtitle">Construction of the expression plasmids</h6>
                    <p class="content_context">
                        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.
                    </p>
                    <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>
                    <p class="content_context" style="text-align:center; font-size:18px">
                        Figure. 1 Schematic diagram of construction of mulita-functional plasmids.
                    </p>

                    <!------------------------part 2------------------------------ -->
                    <h6 class="content_sub_subtitle">Design and integration of three different modules (gshF, met K
                        and cwaA)</h6>
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                        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.
                    </p>
                    <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>
                    <p class="content_context" style="text-align:center; font-size:18px">
                        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.
                    </p>

                    <!------------------------part 3------------------------------ -->
                    <h6 class="content_sub_subtitle">SDS-PAGE detection of protein GshF and MetK</h6>
                    <p class="content_context">
                        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 <i>L.lactis</i> 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.
                    </p>
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                        Figure. 3 SDS PAGE validation of gene gshF and metK expression in <i>L.lactis</i>. M represented
                        marker. WT represented <i>L.lactis</i> NZ9000. 1-3 represented <i>L.lactis</i>/pNZ-GMcA induced with 100,
                        50 and 20 ng/ml nisin.
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