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

 
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            <h1 style="color:white; font-size:80px; padding-top:120px;">Demonstration</h1>
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    <div class="title text-center">
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        <h1 style="color:white; font-size:80px; padding-top:120px;">Demonstration</h1>
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    </div>
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<div class="container">
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    <div class="para">
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            <p style="font-size:30px">For the whole circuit construct, we did following experiments:</p>
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            <h2>1. Gel electrophoresis</h2>
 
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         <div class="fig" align="center">
 
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             <figure>
 
             <figure>
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                 <img width="650px" src="https://static.igem.org/mediawiki/2017/3/30/Demonstration_demo.png">
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                 <img width="650px" src="https://static.igem.org/mediawiki/2017/5/55/Gel_electrophoresis_E.jpeg">
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                <figcaption>
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                </figcaption>
 
             </figure>
 
             </figure>
 
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            <h2>2. Expression of LL-37 in bacteria screened by dot blotting</h2>
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        </div>
  
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        <div class="fig" align="center">
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            <figure>
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                <img width="650px" src="https://static.igem.org/mediawiki/2017/7/7a/Demonstration_9.png">
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            </figure>
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      <figcaption>
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                    <strong>Fig. 2: Result of dot blot. The left light ring shows the protein was produced and the right dark region stands for the non-induced (negative control). The result shows that the whole circuit can be induced by IPTG
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</strong>
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                </figcaption>
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        </div>
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        <div class="para">
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                <h2>3. Inhibition Discs</h2>
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                <p style="font-size:20px">This experiment was to qualitatively evaluate the effectivity of Anti-Microbial Peptides (AMPs). </p>
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            </div>
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        <div class="fig" align="center">
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                <figure class="col-sm-6">
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                    <img height="350px" src="https://static.igem.org/mediawiki/2017/0/0e/Inhibition_ringA.png">
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                    <figcaption><strong>A</strong></figcaption>
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                </figure>
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                <figure class="col-sm-6">
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                    <img height="350px" src="https://static.igem.org/mediawiki/2017/c/c3/Inhibition_ringB.png">
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                    <figcaption><strong>B</strong></figcaption>
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                </figure>
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            </div>
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            <div class="fig" align="center">
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                    <figure class="col-sm-6">
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                        <img height="350px" src="https://static.igem.org/mediawiki/2017/6/63/Inhibition_ringC.png">
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                        <figcaption><strong>C</strong></figcaption>
 +
                    </figure>
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                    <figure class="col-sm-6">
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                        <img height="350px" src="https://static.igem.org/mediawiki/2017/9/9a/Inhibition_ringD.png">
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                        <figcaption><strong>D</strong></figcaption>
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                    </figure>
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                    <figcaption><strong>Fig. 3: Inhibition discs of anti-microbial peptides on Staphylococcus aureus. The concentration of peptides is 1mg/ml and volume is 20ul. (A) LL-37 (B) Grammistin-Pp1 (C) GF-17 (D) three peptides (LL-37, Grammistin-Pp1, and GF-17) mix.</strong></figcaption>
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                </div>
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<div class="para">
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                        <p style="font-size:20px">Fig. 3 shows the AMPs have effectivity on <i>Staphylococcus aureus</i>.</p>
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                </div>
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</div>
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<div class="container">
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        <div class="para">
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            <p style="font-size:30px">
 +
                Aimed at examining the efficacy of the antimicrobial peptides on killing S. aureus, each experiment below were repeated 3
 +
                times.
 +
            </p>
 +
 +
            <h2>4. Determination of the minimal bactericidal concentration (MBC) of the three AMPs.</h2>
 +
        </div>
 +
        <div class="fig" align="center">
 +
            <figure class="col-sm-6">
 +
                <img height="350px" src="https://static.igem.org/mediawiki/2017/6/6f/LL37_MIC.jpeg">
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            </figure>
 +
            <figure class="col-sm-6">
 +
                <img height="350px" src="https://static.igem.org/mediawiki/2017/d/d6/GF-17_MIC.jpeg">
 +
            </figure>
 +
        </div>
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        <div class="fig" align="center">
 +
            <figure class="col-sm-6">
 +
                <img height="350px" src="https://static.igem.org/mediawiki/2017/6/67/Grammistin-Pp1_MIC.jpeg">
 +
            </figure>
 +
            <figure class="col-sm-6">
 +
                <img height="350px" src="https://static.igem.org/mediawiki/2017/5/5e/AMPs_MIC.jpeg">
 +
            </figure>
 +
        </div>
 +
 +
        <div class="para">
 +
            <p style="font-size:20px">
 +
                Both single peptide of the three AMPs and the mixture of them had conspicuous lethal effect on S. aureus.
 +
        </div>
 +
 +
        <div class="para">
 +
            <h2>5. Absorbance (cell density) of the biofilm of
 +
                <i>S. aureus</i> against different concentrations of the three individual AMPs and a mixture of them </h2>
 +
        </div>
  
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                 <img width="650px" src="https://static.igem.org/mediawiki/parts/6/6c/LL-37_biofilm_XJTLU-CHINA.jpeg">
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                 <img width="650px" src="https://static.igem.org/mediawiki/2017/d/da/LL-37_Abs.jpeg">
 
             </figure>
 
             </figure>
 +
            <figure>
 +
                <img width="650px" src="https://static.igem.org/mediawiki/2017/c/c4/GF-17_Abs.jpeg">
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            </figure>
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            <figure>
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                <img width="650px" src="https://static.igem.org/mediawiki/2017/a/af/Grammistin-Pp1_Abs.jpeg">
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            </figure>
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            <figure>
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                <img width="650px" src="https://static.igem.org/mediawiki/2017/7/7b/AMPs_Abs.jpeg">
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            </figure>
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        </div>
 +
 +
        <div class="para">
 +
            <p style="font-size:20px">The biofilm formation of S. aureus was to a great extent inhibited by these three AMPs.</p>
 +
        </div>
 +
 +
        <div class="para">
 +
            <h1>Experience and conclusions:</h1>
 +
            <ul>
 +
                <li>Peptides has shown their antimicrobial activity on
 +
                    <i>S. aureus</i>. And they can successfully expressed in
 +
                    <i>E. coli. </i>
 +
                </li>
 +
                <li>
 +
                    AgrA, which is an essential protein in quorum sensing construct, signifies toxicity to cells during growth.
 +
                </li>
 +
                <li>
 +
                    We have trouble in obtaining enough plasmid DNA extracted from
 +
                    <i>L. lactis </i>after transformation, probably due to either low copy number or low yield from the extraction
 +
                    protocol.
 +
                </li>
 +
            </ul>
 +
        </div>
 +
    </div>
 +
 +
    <!-- banner -->
 +
    <div class="text-center" style="background:#cfe5d9;">
 +
            <h1 style="font-weight:bolder;color:#006934; ">Collaborators and Supporters</h1>
 +
            <div class="container-fluid supporters-logos">
 +
            <div class="row" style="padding-bottom:10px">
 +
           
 +
                <div class="col-md-4 col-sm-6">
 +
                    <a href="http://www.synbio-tech.com.cn"><img class="" src="https://static.igem.org/mediawiki/2017/3/38/Synbio_tech_logo.png"></a>
 +
                </div>
 +
               
 +
                <div class="col-md-4 col-sm-6">
 +
                    <a href="http://www.wx2h.com/web/index.php"><img src="https://static.igem.org/mediawiki/2017/f/f7/Wuxi_no2_hospital_logo.png"></a>
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                </div>
 +
               
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                <div class="col-md-4 col-sm-6">
 +
                    <a href="http://www.chinapeptides.qianyan.biz"><img src="https://static.igem.org/mediawiki/2017/7/7d/Qiangyao_logo.png"></a>
 +
                </div>
 +
           
 +
                <div class="col-md-4 col-sm-6"> 
 +
                    <a href="https://www.neb.com"><img class="" src="https://static.igem.org/mediawiki/2017/0/06/NEB_logo.png"></a>
 +
                </div>
 +
               
 +
                <div class="col-md-4 col-sm-6">
 +
                    <a href="https://www.snapgene.com"><img src="https://static.igem.org/mediawiki/2017/c/cb/Snapgene_logo.png"></a>
 +
                </div>
 +
               
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                <div class="col-md-4 col-sm-6">
 +
                    <a href="http://www.genscript.com"><img src="https://static.igem.org/mediawiki/2017/9/9b/Genscript.png"></a>
 +
                </div>
 +
            </div>
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            </div><!-- /.container --!>
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            </div><!-- /.text-center --!>
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 +
            <!-- footer -->
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            <footer>
 +
            <div class="text-center">
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                <div class="container-fluid">
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                    <div class="row">
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                      <div class="col-md-4 loaction">
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                          <h4>Location</h4>
 +
                          <p  style="text-align:center;">Rm 363, Science Building<br>
 +
                          Xi'an Jiaotong-Liverpool University<br>
 +
                          111 Ren'ai Road, Suzhou, China<br>
 +
                          215123
 +
                          </p>
 +
                      </div>
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                      <div class="col-md-4 social">
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                          <h4>Social</h4>
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                          <a href=""><img src="https://static.igem.org/mediawiki/2017/9/9f/XJTLU_facebook.png" alt="facebook" width=30 height=30></a>
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                          <a href=""><img src="https://static.igem.org/mediawiki/2017/7/72/XJTLU_blog.png" alt="blog" width=30 height=30></a>
 +
                      </div>
 +
                      <div class="col-md-4 contact">
 +
                          <h4>Get in touch</h4>
 +
                          <img src="https://static.igem.org/mediawiki/2017/1/19/XJTLU_email.png" alt="emali" width=30 height=30>
 +
                          <p style="text-align:center;">igem@xjtlu.edu.cn</p>
 +
                      </div>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
            <div class="text-center" style="background:#003b73;">
 +
                    <p style="text-align:center; color:white;">XJTLU-CHINA iGEM 2017</p>
 +
            </div>
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            </footer>
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</body>
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</html>

Latest revision as of 13:20, 1 December 2017

Desmonstration

Demonstration

For the whole circuit construct, we did following experiments:

1. Gel electrophoresis

2. Expression of LL-37 in bacteria screened by dot blotting

Fig. 2: Result of dot blot. The left light ring shows the protein was produced and the right dark region stands for the non-induced (negative control). The result shows that the whole circuit can be induced by IPTG

3. Inhibition Discs

This experiment was to qualitatively evaluate the effectivity of Anti-Microbial Peptides (AMPs).

A
B
C
D
Fig. 3: Inhibition discs of anti-microbial peptides on Staphylococcus aureus. The concentration of peptides is 1mg/ml and volume is 20ul. (A) LL-37 (B) Grammistin-Pp1 (C) GF-17 (D) three peptides (LL-37, Grammistin-Pp1, and GF-17) mix.

Fig. 3 shows the AMPs have effectivity on Staphylococcus aureus.

Aimed at examining the efficacy of the antimicrobial peptides on killing S. aureus, each experiment below were repeated 3 times.

4. Determination of the minimal bactericidal concentration (MBC) of the three AMPs.

Both single peptide of the three AMPs and the mixture of them had conspicuous lethal effect on S. aureus.

5. Absorbance (cell density) of the biofilm of S. aureus against different concentrations of the three individual AMPs and a mixture of them

The biofilm formation of S. aureus was to a great extent inhibited by these three AMPs.

Experience and conclusions:

  • Peptides has shown their antimicrobial activity on S. aureus. And they can successfully expressed in E. coli.
  • AgrA, which is an essential protein in quorum sensing construct, signifies toxicity to cells during growth.
  • We have trouble in obtaining enough plasmid DNA extracted from L. lactis after transformation, probably due to either low copy number or low yield from the extraction protocol.

Collaborators and Supporters

Location

Rm 363, Science Building
Xi'an Jiaotong-Liverpool University
111 Ren'ai Road, Suzhou, China
215123

Get in touch

emali

igem@xjtlu.edu.cn

XJTLU-CHINA iGEM 2017