Difference between revisions of "Team:XJTLU-CHINA/Demonstrate"

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<div class="column full_size">
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        .front,
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<h1>Demonstrate</h1>
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        .back {
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<h3>Gold Medal Criterion #4</h3>
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<p>
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        .front {
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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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<p>
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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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<body style="background-color: rgb(84,146,197);">
  
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</div>
 
  
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<div class="container-fluid" style="background-color: rgb(84,146,197); padding: 40px 30px; margin-top: 35px">
  
  
  
 +
    <div style="margin-top: 20px; width: 100px; float: left"></div>
 +
    <img src="https://static.igem.org/mediawiki/2018/1/1a/T--XJTLU-CHINA--lo.png" style="width: 140px; margin-top: 20px; float: left">
 +
    <h1 style="font-family:'Impact'; color: white; font-size: 80px; margin-top: 20px; margin-bottom: 50px; float: left; margin-left: 20px">
 +
        Demonstration
 +
    </h1>
  
 +
    <div class="container-fluid" style="background-color: white; box-shadow: 0px 0px 4px rgba(0,0,0,0.56); border-radius: 5px; margin-top: 200px; padding-bottom: 50px">
 +
 +
        <div style="text-align: center; margin-top: 50px; border-bottom: 0.8px solid #337ab7; padding-bottom: 20px">
 +
            <h1 style="font-family:'Impact'; color: #337ab7">
 +
                Exosome production boosting
 +
            </h1>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 20px">
 +
            <div class="col-xs-1"></div>
 +
            <div class="col-xs-10">
 +
                <h2 style="font-family:'Impact'; color: #337ab7; margin-bottom: 20px">Experiments</h2>
 +
                <h3 style="font-family:'Impact'; color: #337ab7; margin-bottom: 20px">1. Exosomal biomarker synthesis</h3>
 +
                <p style="font-size: 17px; text-align: justify; font-weight: normal; margin-top: 20px; margin-bottom: 20px">
 +
                    We transfected HEK293T cells with nanoluciferase coding sequence fused with exosomal biomarker membrane protein CD63 (BBa_K2619100) for the subsequent quantification of exosomes.
 +
                </p>
 +
            </div>
 +
            <div class="col-xs-1"></div>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 10px">
 +
            <div class="col-xs-2"></div>
 +
            <div class="col-xs-8" style="text-align: center">
 +
                <img src="https://static.igem.org/mediawiki/2018/0/09/T--XJTLU-CHINA--demo1.png" alt="..." class="img-thumbnail" style="width: 80%">
 +
                <p style="text-align: center"><strong>Figure 1: Negative control was done by transfecting the HEK293T cells with empty pcDNA3.1 plasmid</strong></p>
 +
            </div>
 +
            <div class="col-xs-2"></div>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 20px">
 +
            <div class="col-xs-1"></div>
 +
            <div class="col-xs-10">
 +
                <h3 style="font-family:'Impact'; color: #337ab7; margin-bottom: 20px">2. Exosome boosting analysis by luciferase assay</h3>
 +
                <p style="font-size: 17px; text-align: justify; font-weight: normal; margin-top: 20px; margin-bottom: 20px">
 +
                    Since the biomarker of exosomes was synthesized successfully in experiment 1.
 +
                    We then conducted experiments to test the exosome production boosting ability of our deigned devices (BBa_K2619014—nSMase and BBa_K2619103—Booster) by co-transfection.
 +
                </p>
 +
            </div>
 +
            <div class="col-xs-1"></div>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 10px">
 +
            <div class="col-xs-2"></div>
 +
            <div class="col-xs-8" style="text-align: center">
 +
                <img src="https://static.igem.org/mediawiki/2018/c/cb/T--XJTLU-CHINA--demo2.png" alt="..." class="img-thumbnail" style="width: 80%">
 +
                <p style="text-align: center">
 +
                    <strong>
 +
                        Figure 2: NC: negative control;
 +
                        done by transfecting CD63-nluc and empty pcDNA3.1 plasmid (1:2 ratio).
 +
                        Booster: Steap-SDC4-NadB (BBa_2619103); done by transfecting CD63-nluc,
 +
                        Booster and pcDNA3.1 plasmid (1:1:1 ratio). Booster nSMase: done by transfecting Booster,
 +
                        nSMase and CD63-nluc (1:1:1 ratio)
 +
                    </strong>
 +
                </p>
 +
            </div>
 +
            <div class="col-xs-2"></div>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 20px">
 +
            <div class="col-xs-1"></div>
 +
            <div class="col-xs-10">
 +
                <h3 style="font-family:'Impact'; color: #337ab7; margin-bottom: 20px">
 +
                    3. Exosome production boosting analysis by Pierce BCA protein assay
 +
                </h3>
 +
                <p style="font-size: 17px; text-align: justify; font-weight: normal; margin-top: 20px; margin-bottom: 20px">
 +
                    Our team utilized varied methods to confirm the validity of exosomal production enhancement of Booster and nSMase.
 +
                </p>
 +
            </div>
 +
            <div class="col-xs-1"></div>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 10px">
 +
            <div class="col-xs-2"></div>
 +
            <div class="col-xs-8" style="text-align: center">
 +
                <img src="https://static.igem.org/mediawiki/2018/4/41/T--XJTLU-CHINA--demo3.png" alt="..." class="img-thumbnail" style="width: 80%">
 +
                <p style="text-align: center">
 +
                    <strong>
 +
                        Figure 3: NC: negative control; done by transfecting empty pcDNA3.1 plasmid.
 +
                        Booster: Steap-SDC4-NadB; done by transfecting Booster and pcDNA3.1 plasmid (1:1 ratio).
 +
                        Booster nSMase: done by transfecting Booster and nSMase (1:1 ratio)
 +
                    </strong>
 +
                </p>
 +
            </div>
 +
            <div class="col-xs-2"></div>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 20px">
 +
            <div class="col-xs-1"></div>
 +
            <div class="col-xs-10">
 +
                <h3 style="font-family:'Impact'; color: #337ab7; margin-bottom: 20px">
 +
                    4. Exosome production boosting analysis by nanoparticle tracking assay (NTA)
 +
                </h3>
 +
                <p style="font-size: 17px; text-align: justify; font-weight: normal; margin-top: 20px; margin-bottom: 20px">
 +
                    The third method of verifying the devices capacity in exosome production enhancement is doing NTA by NanosightTM.
 +
                    After purification of exosomes by Total Exosome Isolation Reagent (Thermo ScientificTM),
 +
                    the exact secreted particle numbers were analyzed.
 +
                </p>
 +
            </div>
 +
            <div class="col-xs-1"></div>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 10px">
 +
            <div class="col-xs-2"></div>
 +
            <div class="col-xs-8" style="text-align: center">
 +
                <img src="https://static.igem.org/mediawiki/2018/6/64/T--XJTLU-CHINA--demo4.png" alt="..." class="img-thumbnail" style="width: 80%">
 +
                <p style="text-align: center">
 +
                    <strong>
 +
                        Figure 4: NC: negative control; done by transfecting empty pcDNA3.1 plasmid.
 +
                        Booster: Steap-SDC4-NadB; done by transfecting Booster and pcDNA3.1 plasmid (1:1 ratio).
 +
                        Booster nSMase: done by transfecting Booster and nSMase (1:1 ratio)
 +
                    </strong>
 +
                </p>
 +
            </div>
 +
            <div class="col-xs-2"></div>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 20px">
 +
            <div class="col-xs-1"></div>
 +
            <div class="col-xs-10">
 +
                <h2 style="font-family:'Impact'; color: #337ab7; margin-bottom: 20px">
 +
                    Conclusion
 +
                </h2>
 +
                <p style="font-size: 17px; text-align: justify; font-weight: normal; margin-top: 20px; margin-bottom: 20px">
 +
                    A combination of two exosome production accelerators/enhancing devices is reported to be the most capable of boosting the cells in exosome production.
 +
                </p>
 +
            </div>
 +
            <div class="col-xs-1"></div>
 +
        </div>
 +
 +
        <div style="text-align: center; margin-top: 50px; border-bottom: 0.8px solid #337ab7; padding-bottom: 20px">
 +
            <h1 style="font-family:'Impact'; color: #337ab7">
 +
                Neuronal cell targeting experiments
 +
            </h1>
 +
        </div>
 +
 +
        <div class="row" style="margin-top: 20px">
 +
            <div class="col-xs-1"></div>
 +
            <div class="col-xs-10">
 +
                <h2 style="font-family:'Impact'; color: #337ab7; margin-bottom: 20px">
 +
                    Lamp2b protein linkage experiment
 +
                </h2>
 +
                <p style="font-size: 17px; text-align: justify; font-weight: normal; margin-top: 20px; margin-bottom: 20px">
 +
                    We fused nanoluciferase gene with exosomal membrane protein Lamp2b to examine its downstream gene expression. (Lamp2b-nluc, BBa_K2619113).
 +
                </p>
 +
            </div>
 +
            <div class="col-xs-1"></div>
 +
        </div>
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 +
        <div class="row" style="margin-top: 10px; margin-bottom: 40px">
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            <div class="col-xs-2"></div>
 +
            <div class="col-xs-8" style="text-align: center">
 +
                <img src="https://static.igem.org/mediawiki/2018/4/47/T--XJTLU-CHINA--demo5.png" alt="..." class="img-thumbnail" style="width: 80%">
 +
                <p style="text-align: center">
 +
                    <strong>
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                        Figure 5: NC: negative control; done by transfecting empty pcDNA3.1 plasmid.
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Latest revision as of 01:06, 18 October 2018

Demonstration

Exosome production boosting

Experiments

1. Exosomal biomarker synthesis

We transfected HEK293T cells with nanoluciferase coding sequence fused with exosomal biomarker membrane protein CD63 (BBa_K2619100) for the subsequent quantification of exosomes.

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Figure 1: Negative control was done by transfecting the HEK293T cells with empty pcDNA3.1 plasmid

2. Exosome boosting analysis by luciferase assay

Since the biomarker of exosomes was synthesized successfully in experiment 1. We then conducted experiments to test the exosome production boosting ability of our deigned devices (BBa_K2619014—nSMase and BBa_K2619103—Booster) by co-transfection.

...

Figure 2: NC: negative control; done by transfecting CD63-nluc and empty pcDNA3.1 plasmid (1:2 ratio). Booster: Steap-SDC4-NadB (BBa_2619103); done by transfecting CD63-nluc, Booster and pcDNA3.1 plasmid (1:1:1 ratio). Booster nSMase: done by transfecting Booster, nSMase and CD63-nluc (1:1:1 ratio)

3. Exosome production boosting analysis by Pierce BCA protein assay

Our team utilized varied methods to confirm the validity of exosomal production enhancement of Booster and nSMase.

...

Figure 3: NC: negative control; done by transfecting empty pcDNA3.1 plasmid. Booster: Steap-SDC4-NadB; done by transfecting Booster and pcDNA3.1 plasmid (1:1 ratio). Booster nSMase: done by transfecting Booster and nSMase (1:1 ratio)

4. Exosome production boosting analysis by nanoparticle tracking assay (NTA)

The third method of verifying the devices capacity in exosome production enhancement is doing NTA by NanosightTM. After purification of exosomes by Total Exosome Isolation Reagent (Thermo ScientificTM), the exact secreted particle numbers were analyzed.

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Figure 4: NC: negative control; done by transfecting empty pcDNA3.1 plasmid. Booster: Steap-SDC4-NadB; done by transfecting Booster and pcDNA3.1 plasmid (1:1 ratio). Booster nSMase: done by transfecting Booster and nSMase (1:1 ratio)

Conclusion

A combination of two exosome production accelerators/enhancing devices is reported to be the most capable of boosting the cells in exosome production.

Neuronal cell targeting experiments

Lamp2b protein linkage experiment

We fused nanoluciferase gene with exosomal membrane protein Lamp2b to examine its downstream gene expression. (Lamp2b-nluc, BBa_K2619113).

...

Figure 5: NC: negative control; done by transfecting empty pcDNA3.1 plasmid.

Collaborators and Supporters

Location

Rm 363, Science Building

Xi'an Jiaotong-Liverpool University

111 Ren'ai Road, Suzhou, China

215123

Social

FB

Get in touch

igem@xjtlu.edu.cn