Difference between revisions of "Team:NKU China/Parts"

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             //particles();

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         <table>

             <caption>Part Submissions</caption>

            <tr>

                <td>Biobrick</td>

                <td>Part</td>

                <td>Description</td>

            </tr>

            <tr>

                <td>BBa_K1981001</td>

                <td><i>lsrA</i></td>

                <td>Autoinducer 2 import ATP-binding protein LsrA</td>

            </tr>

            <tr>

                <td>BBa_K1981002</td>

                <td><i>lsrB</i></td>

                <td>Autoinducer 2-binding protein LsrB</td>

            </tr>

            <tr>

                <td>BBa_K1981003</td>

                <td><i>lsrC</i></td>

                <td>Autoinducer 2 import system permease protein LsrC</td>

            </tr>

            <tr>

                <td>BBa_K1981004</td>

                <td><i>lsrD</i></td>

                <td>Autoinducer 2 import system permease protein LsrD</td>

            </tr>

            <tr>

                <td>BBa_K1981005</td>

                <td><i>lsrFG</i></td>

                <td>A Protein complex involved in the degradation of phospho-AI-2</td>

            </tr>

            <tr>

                <td>BBa_K1981006</td>

                <td><i>lsrK</i></td>

                <td>Autoinducer-2 kinase</td>

            </tr>

            <tr>

                <td>BBa_K1981007</td>

                <td><i>luxS</i></td>

                <td>S-ribosylhomocysteine lyase</td>

            </tr>

            <tr>

                <td>BBa_K1981008</td>

                <td><i>Mtn</i></td>

                <td>5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase</td>

            </tr>

            <tr>

                <td>BBa_K1981101</td>

                <td><i>plsr</i></td>

                <td><i>plsr</i> is the promoter of the <i>lsr</i> operon</td>

            </tr>

            <tr>

                <td>BBa_K1981201</td>

                <td>Autoinducer-2 Response Device A</td>

                <td>A composite part of which GFP expression  can directly respond to AI-2 concentration in the nature or artificial environment</td>

            </tr>

            <tr>

                <td>BBa_K1981202</td>

                <td>Autoinducer-2 Response Device B</td>

                <td>A composite part which has a tighter regulation and delayed response compared to AI-2 response device A</td>

            </tr>

        </table>

                 <div class="p">AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by E.coli MG1655, AI-2 response involves lsr gene clusters that encode lsrACDB, lsrK, lsrFG. plsr is the promoter of the lsr operon.</div>

                 <div class="p">We isolated the promoter region of the lsr operon to regulate the expression of the report gene, GFP (BBa_E0040). In order to test the function of the lsr promoter, we transformed the plasmid pTrcHisB containing plsr with GFP gene at its downstream into E. coli MG1655 delta luxS. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50&mu;M. Every one hour, optical density was measured and samples were harvested for HPLC analysis. As you can see, after induced by AI-2, the GFP expression is increased compared to control group.</div>

                 <figure>

                     <figcaption>Figure 1.1: GFP expression after promoter lsr is induced by exogenous AI-2</figcaption>

                 <div class="p">Then we test whether promoter lsr can respond to different AI-2 concentration. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50&mu;M, 40&mu;M, 30&mu;M, 20&mu;M, 10&mu;M, 0&mu;M. Every one hour, optical density was measured and samples were harvested for HPLC analysis. The result below shows that promoter lsr can respond to different AI-2 concentration resulting in different GFP expression.</div>

                 <figure>

                 <div class="p">This composite part consists of the AI-2 (autoinducer-2) quorum sensor-inducible promoter BBa_K1981101, a GFP coding sequence BBa_E0040, a double terminator BBa_B0015. We firstly isolated promoter lsr from E.coli MG1655. GFP BBa_E0040 and double terminator BBa_B0015 are standard part offered by iGEM. Then we successfully constrcuted plsr+ GFP +double terminator by using homologous recombination technology.</div>

                 <div class="p">In AI-2 Response Device A, GFP expression is under the control of promoter, lsr. When phospho-AI-2 binds LsrR, expression of GFP ensues. The expression of GFP can directly response to the AI-2 level in the environment, which is an alternative way to reflect the AI-2 concentration in the nature or artificial environment.</div>

                 <figure>

                 <div class="p">AI-2 Consumers was constructed by iGEM 2016 NKU_China by overexpression the components responsible for AI-2 uptake (lsrACDB), phosphorylation (lsrK), and degradation (lsrFG), which can directly absorb and degrade AI-2 in the nature or artificial environment. When E.coli consisting of AI-2 Response Device A are co-cultured with AI-2 Consumers, the GFP expression of AI-2 Response Device A is directly decreased compared to control group.</div>

                 <figure>

                     <figcaption>Figure 1.2: GFP expression of AI-2 Response Device A when co-cultured with AI-2 Consumers.</figcaption>

                 <div class="p">AI-2 Suppliers was constructed by iGEM 2016 NKU_China by overexpression the components responsible for AI-2 production (luxS, mtn), which can directly supply and enrich the AI-2 molecular level in the nature or artificial environment. When E.coli consisting of AI-2 Response Device A are co-cultured with AI-2 Suppliers, the GFP expression of AI-2 Response Device A is directly increased compared to control group.</div>

                 <figure>