Difference between revisions of "Team:TJU China/Demonstrate"

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<h1>Demonstrate</h1>
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<h3>Gold Medal Criterion #4</h3>
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve their gold medals!
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Please see the <a href="https://2018.igem.org/Judging/Medals">2018 Medals Page</a> for more information.
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                        <a href="https://2018.igem.org/Team:TJU_China/Demonstrate">Demonstrate</a>
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        <div class="head">Enhanced sensitivity of metal iron detection based on dCas9 system</div>
 +
        <div class="content">In view of the current serious pollution problems, we focus on the pollution of heavy metal ions. Only by detecting
 +
            heavy metal ions quickly and accurately can we prevent pollution in a timely manner. To this end, we started
 +
            with a arsenic ion, combined with the achievements of the 2006 iGEM team (iGEM2006_Edinburgh) in order to construct
 +
            a circuit dedicated to the detection of arsenic ions, which consists of Promoter J23104, ArsR Protein, Promoter
 +
            ArsR, smURFP. We first ligated these fragments by overlap, then ligated them to the pKM586 plasmid by double
 +
            restriction enzymes, and then transformed them into E.coli BL21. Since we think this loop can not be completed
 +
            to meet our requirements, we want to make this loop more sensitive. Therefore, we noticed that dcas9 in the CRISPR-Cas
 +
            system has an enhanced transcriptional effect, thus amplifying the effect of arsenic ions on the loop. In the
 +
            plasmid of dCas9, we need to cut the two segments of the plasmid with BsaI enzyme, then connect the spacer we
 +
            designed to target dCas9 to the corresponding gene, and then we import it with another plasmid E.coli BL21 to
 +
            complete the enhancement of our arsenic sensing circuit by dCas9.</div>
 +
        <div>
 +
            <img src="https://static.igem.org/mediawiki/2018/5/5b/T--TJU_China--d1.1.png">
 +
        </div>
 +
        <div class="figure"><b>Figure1:</b>The result of nucleic acid gel electrophoresis of Bba-J33201 after PCR. Lane M, Marker. Lane 1-6，Bba-J33201</div>
 +
        <div><img src="https://static.igem.org/mediawiki/2018/d/d1/T--TJU_China--d1.2.png"></div>
 +
        <div class="figure"><b>Figure2:</b>The result of nucleic acid gel electrophoresis of smURFP after PCR.LaneM, Marker. Lane1-8, smURFP</div>
 +
        <div><img src="https://static.igem.org/mediawiki/2018/6/66/T--TJU_China--d1.3.png"></div>
 +
        <div class="figure"><b>Figure3:</b>The result of nucleic acid gel electrophoresis after overlapping of J23104 and ArsR Protein. LaneM, Marker. Lane 1，ArsR Promoter；Lane 2-5：J23104＋ArsR Protein.</div>
 +
        <div><img src="https://static.igem.org/mediawiki/2018/5/5e/T--TJU_China--d1.4.png"></div>
 +
        <div class="figure"><b>Figure4:</b>The result of nucleic acid gel electrophoresis after overlapping of ArsR Promoter and smURFP. LaneM, Marker. Lane 1, smURFP. Lane 2-4,ArsR Promoter＋smURFP</div>
 +
        <div><img src="https://static.igem.org/mediawiki/2018/5/54/T--TJU_China--d1.5.png"></div>
 +
        <div class="figure"><b>Figure5:</b>Double digestion to verify the ligation product. lane M, Marker. Lane 1, Plasmid pKM586. Lane 2, Plasmid pKM586 single digestion with BamHI. Lane 3, Plasmid pKM586 double digestion with AatII and BamHI. Lane 4, Plasmid ArS. Lane 5, Plasmid ArS single digestion with BamHI. Lane 6, Plasmid ArS double gigestion with AatII and BamHI. Lane 7, Plasmid ArS. Lane 8, Plasmid ArS single digestion with BamHI.  Lane 9, Plasmid ArS double digestion with AatII and BamHI.</div>
 +
        <div><img src="https://static.igem.org/mediawiki/2018/3/3a/T--TJU_China--d1.6.png"></div>
 +
        <div class="figure"><b>Figure6:</b>Double digestion of pKM586 with AatII and BamHI. lane M, Marker. Lane 1,Plasmid pKM586. Lane 2, single digestion with BamHI. Lane 3, Plasmid pKM586 after double enzyme digestion</div>
 +
        <div><img src="https://static.igem.org/mediawiki/2018/9/9c/T--TJU_China--d1.7.png"></div>
 +
        <div class="figure"><b>Figure7:</b>Double digestion of pKM586 with AatII and BamHI. LaneM, Marker. Lane 1,Plasmid pKM586. Lane 2, Plasmid pKM586 after double enzyme digestion</div>
 +
 +
    </div>
 +
    <div class="group" id="group2" style="display:none;">
 +
        <div class="head">Targeted delivery of sgRNA/Cas9 complex</div>
 +
        <div>
 +
            <img src="">
 +
        </div>
 +
    </div>
 +
    <div class="group" id="group3" style="display:none;">
 +
        <div class="head">Improved sensitivity of metal iron detection based on dCas9 system</div>
 +
        <div>
 +
            <img src="">
 +
        </div>
 +
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        <div class="figure"><b>Figure.</b></div>
 +
        <div><img src=""></div>
 +
        <div class="figure"><b>Figure.</b></div>
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Revision as of 01:32, 17 October 2018

<!DOCTYPE > home

Enhanced sensitivity of metal iron detection based on dCas9 system
In view of the current serious pollution problems, we focus on the pollution of heavy metal ions. Only by detecting heavy metal ions quickly and accurately can we prevent pollution in a timely manner. To this end, we started with a arsenic ion, combined with the achievements of the 2006 iGEM team (iGEM2006_Edinburgh) in order to construct a circuit dedicated to the detection of arsenic ions, which consists of Promoter J23104, ArsR Protein, Promoter ArsR, smURFP. We first ligated these fragments by overlap, then ligated them to the pKM586 plasmid by double restriction enzymes, and then transformed them into E.coli BL21. Since we think this loop can not be completed to meet our requirements, we want to make this loop more sensitive. Therefore, we noticed that dcas9 in the CRISPR-Cas system has an enhanced transcriptional effect, thus amplifying the effect of arsenic ions on the loop. In the plasmid of dCas9, we need to cut the two segments of the plasmid with BsaI enzyme, then connect the spacer we designed to target dCas9 to the corresponding gene, and then we import it with another plasmid E.coli BL21 to complete the enhancement of our arsenic sensing circuit by dCas9.
Figure1:The result of nucleic acid gel electrophoresis of Bba-J33201 after PCR. Lane M, Marker. Lane 1-6，Bba-J33201
Figure2:The result of nucleic acid gel electrophoresis of smURFP after PCR.LaneM, Marker. Lane1-8, smURFP
Figure3:The result of nucleic acid gel electrophoresis after overlapping of J23104 and ArsR Protein. LaneM, Marker. Lane 1，ArsR Promoter；Lane 2-5：J23104＋ArsR Protein.
Figure4:The result of nucleic acid gel electrophoresis after overlapping of ArsR Promoter and smURFP. LaneM, Marker. Lane 1, smURFP. Lane 2-4,ArsR Promoter＋smURFP
Figure5:Double digestion to verify the ligation product. lane M, Marker. Lane 1, Plasmid pKM586. Lane 2, Plasmid pKM586 single digestion with BamHI. Lane 3, Plasmid pKM586 double digestion with AatII and BamHI. Lane 4, Plasmid ArS. Lane 5, Plasmid ArS single digestion with BamHI. Lane 6, Plasmid ArS double gigestion with AatII and BamHI. Lane 7, Plasmid ArS. Lane 8, Plasmid ArS single digestion with BamHI. Lane 9, Plasmid ArS double digestion with AatII and BamHI.
Figure6:Double digestion of pKM586 with AatII and BamHI. lane M, Marker. Lane 1,Plasmid pKM586. Lane 2, single digestion with BamHI. Lane 3, Plasmid pKM586 after double enzyme digestion
Figure7:Double digestion of pKM586 with AatII and BamHI. LaneM, Marker. Lane 1,Plasmid pKM586. Lane 2, Plasmid pKM586 after double enzyme digestion