Difference between revisions of "Team:NCTU Formosa/Wet Lab"

 
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   <div class="wrapper">
 
   <div class="wrapper">
 
     <div class="banner">
 
     <div class="banner">
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       <img class="cover" src="https://static.igem.org/mediawiki/2018/8/87/T--NCTU_Formosa--Experiment.png">
+
       <img class="cover" src="https://static.igem.org/mediawiki/2018/c/c5/T--NCTU_Formosa--wetlab6.png">
 +
      <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Expression"><img src="https://static.igem.org/mediawiki/2018/6/6e/T--NCTU_Formosa--protein_expression.png" class="protein"></a>
 +
      <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Functional_Analysis"><img src="https://static.igem.org/mediawiki/2018/7/70/T--NCTU_Formosa--MIC.png" class="mic"></a>
 +
      <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Curcumin_Biosensor"><img src="https://static.igem.org/mediawiki/2018/2/20/T--NCTU_Formosa--biosensor.png" class="sensor"></a>
 
     </div>
 
     </div>
−
     <div class="sec1" style="background-color:#e5f1f7;">
+
     <div class="sec1" style="background-color:#ffffff;">
−
       <div class="title"><p>Experiment design</p></div>
+
       <img src="https://static.igem.org/mediawiki/2018/3/30/T--NCTU_Formosa--Experiment_design_title.png" class="title_title">
 
       <div class="text">
 
       <div class="text">
−
         <p>Our system provided a way to regulate microbiota to an ideal balance . Since we focused our model on the excess PSB in soil, which is a large issue of agriculture in Taiwan, we chose antimicrobial peptide as the Bio-stimulator to limit the amount of PSB in soil. To express our target protein, we first designed Biobricks that contain sequences of these peptides. All the experiments we performed with Biobricks are mentioned below.
+
         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Our system provided a way to regulate microbiota to an ideal balance. Since we focused our model on the excess phosphate solubilizing bacteria(PSB) in soil, which is a large issue of agriculture in Taiwan, we chose antimicrobial peptide as the bio-stimulator to limit the amount of PSB in soil. To express our target protein, we first designed BioBricks that contain sequences of these peptides. All the experiments we performed with BioBricks are mentioned below.
 
</p>
 
</p>
 
       </div>
 
       </div>
 
     </div>
 
     </div>
 
     <div class="sec2" style="background-color:#FFFFFF;">
 
     <div class="sec2" style="background-color:#FFFFFF;">
−
       <div class="title"><p>BioBrick</p></div>
+
       <div class="title_1" style="margin-top: 0;"><p>BioBrick</p></div>
 
       <div class="text">
 
       <div class="text">
−
         <p>The BioBrick we designed contains a T7 promoter, induced by IPTG, and RBS with our target protein behind. We also added a intein-CBD (chitin binding domain) tag behind bacteriocin to better purify our peptide.</p>
+
         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The BioBrick we designed contains a T7 promoter, induced by IPTG, and RBS with our target protein behind. We also added a gene fragment of intein-CBD (chitin binding domain) tag behind the bacteriocin gene to better purify our peptide.</p>
 
       </div>
 
       </div>
−
       <img src="https://static.igem.org/mediawiki/2018/a/aa/T--NCTU_Formosa--biobrick.png" class="expression" style="display:block; margin:auto;" width="700">
+
       <img src="https://static.igem.org/mediawiki/2018/a/aa/T--NCTU_Formosa--biobrick.png" class="expression" style="display:block; margin:auto; margin-bottom: 1vw;" width="60%">
 
       <div class="explanation">
 
       <div class="explanation">
 
         <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
 
         <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
−
         Figure 1. BioBrick: T7 promoter + RBS + target peptide + intein-CBD
+
         Figure 1: BioBrick: T7 promoter + RBS + target peptide + intein-CBD.
 
       </div>
 
       </div>
 +
      <div class="title_1"><p>Bacteriocin</p></div>
 
       <div class="text">
 
       <div class="text">
−
         <p>Intein is a protein segment that is able to excise itself from the larger protein it binds to. Therefore, it is also called “ protein introns”. We utilized intein-CBD tag to purify our peptides.
+
         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;It is a peptidic toxin produced by bacteria that inhibits the growth of similar or closely related bacterial strains, but does not damage the bacteria themselve by specific immune proteins. It is heat stable and shows its antimicrobial activity in various temperatures and pH levels. Unlike most antibiotics, which are secondary metabolites, bacteriocins are ribosomally synthesized and sensitive to proteases while generally harmless to the human body and surrounding environment.</p>
−
Although affinity tags have been widely used to purify recombinant proteins, it must removed by protease in the final purification. In contrast, intein-CBD tag will undergo the cleavage reaction with DTT (1,4-dithiothreitol) or cysteine, which will not cause the structure changes of our short peptide.
+
        <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Most bacteriocins interact with anionic lipids that are abundantly present in the membranes of gram-positive bacteria. The receptors on the target membrane can determine the specificity of bacteriocins, and the insertion of bacteriocins is driven by proton motive force.</p>
−
</p>
+
 
       </div>
 
       </div>
 +
      <img src="https://static.igem.org/mediawiki/2018/b/bc/T--NCTU_Formosa--mechanism.png" class="expression" style="display:block; margin:auto; margin-bottom: 1vw;" width="25%">
 
       <div class="explanation">
 
       <div class="explanation">
 
         <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
 
         <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
−
         Figure 2. Procedure of intein-mediated protein splicing
+
         Figure 2: Mechanism of bacteriocin.
 
       </div>
 
       </div>
−
       <div class="title_1"><p>1. Protein expression</p></div>
+
       <div class="title_1"><p>Purification</p></div>
−
      <div class="title_2"><p>Cloning:</p></div>
+
 
       <div class="text">
 
       <div class="text">
−
         <p>We cloned the insert gene into pSB1C3 backbone and transform into <i>E. coli</i> DH5α. <a href="https://2018.igem.org/Team:NCTU_Formosa/Parts parts">These</a> have been submitted to iGEM.</p>
+
         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Intein is a protein segment that is able to excise itself from the larger protein it binds to. Therefore, it is also called “ protein introns”. We utilized intein-CBD tag to purify our peptides.<br>&nbsp;&nbsp;&nbsp;&nbsp;Although affinity tags have been widely used to purify recombinant proteins, it must removed by protease in the final purification. In contrast, intein-CBD tag will undergo the cleavage reaction with DTT (1,4-dithiothreitol) or cysteine, which will not cause the structure changes of our short peptide.
 +
        </p>
 
       </div>
 
       </div>
−
       <div class="title_2"><p>Expression:</p></div>
+
       <img src="https://static.igem.org/mediawiki/2018/5/54/T--NCTU_Formosa--purify.png" class="expression" style="display:block; margin:auto; margin-bottom: 1vw;" width="30%">
−
       <div class="text">
+
       <div class="explanation">
−
         <p>We expressed the proteins by <i>E. coli</i> ER2566 and <i>E. coli</i> BL21 Rosetta-Gami.</p>
+
         <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
 +
        Figure 3: Procedure of intein-mediated protein splicing
 +
          </p>
 
       </div>
 
       </div>
−
      <div class="title_2"><p>SDS-PAGE:</p></div>
 
−
      <div class="text">
 
−
        <p>We checked the expression result through SDS-PAGE.</p>
 
−
      </div>
 
−
      <div class="title_1"><p>2. MIC test</p></div>
 
−
      <div class="title_2"><p>Inhibition zone:</p></div>
 
−
      <div class="title_2"><p>Minimum Inhibitory Concentration Broth Microdilution:</p></div>
 
−
      <div class="title_1"><p>3. Growth curve experiment</p></div>
 
−
      <div class="title_1"><p>4. Soil experiment</p></div>
 
 
     </div>
 
     </div>
 +
    <div class="title_1" style="margin-bottom: -19vw; margin-left: 5vw;"><p>Click Icons to<br>see each part!</p></div>
 +
    <div class="bottom">
 +
      <img src="https://static.igem.org/mediawiki/2018/1/10/T--NCTU_Formosa--exp_design2.png" class="bg">
 +
      <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Expression"><img src="https://static.igem.org/mediawiki/2018/9/98/T--NCTU_Formosa--bac_icon.png" class="bac_icon"></a>
 +
      <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Functional_Analysis"><img src="https://static.igem.org/mediawiki/2018/a/ae/T--NCTU_Formosa--inhibition_icon.png" class="inhibition_icon"></a>
 +
      <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Curcumin_Biosensor"><img src="https://static.igem.org/mediawiki/2018/d/d8/T--NCTU_Formosa--curcumin_icon.png" class="curcumin_icon"></a>
 +
      <img src="https://static.igem.org/mediawiki/2018/e/e0/T--NCTU_Formosa--line1.png" class="line1">
 +
      <img src="https://static.igem.org/mediawiki/2018/9/9a/T--NCTU_Formosa--line2.png" class="line2">
 +
      <img src="https://static.igem.org/mediawiki/2018/c/ca/T--NCTU_Formosa--line3.png" class="line3">
 +
      <div class="title" id="title_1"><p>PrOtEin<br>ExprEssiOn</p></div>
 +
      <div class="title" id="title_2"><p>FunctiOnAl<br>AnAlysis</p></div>
 +
      <div class="title" id="title_3"><p>Curcumin<br>BiO-sEnsOr</p></div>
 +
      <div class="intro" id="intro_1"><p>We expressed our proteins by <i>E. coli</i> BL21 Rosetta-gami.</p></div>
 +
      <div class="intro" id="intro_2"><p>We checked the inhibitory activity of our bio-stimulator, bacteriocins. The analysis included inhibition zone and inhibition ability.</p></div>
 +
      <div class="intro" id="intro_3"><p>The detection of curcumin can perfect our productivity model.</p></div>
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Latest revision as of 01:31, 18 October 2018

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     Our system provided a way to regulate microbiota to an ideal balance. Since we focused our model on the excess phosphate solubilizing bacteria(PSB) in soil, which is a large issue of agriculture in Taiwan, we chose antimicrobial peptide as the bio-stimulator to limit the amount of PSB in soil. To express our target protein, we first designed BioBricks that contain sequences of these peptides. All the experiments we performed with BioBricks are mentioned below.

BioBrick

     The BioBrick we designed contains a T7 promoter, induced by IPTG, and RBS with our target protein behind. We also added a gene fragment of intein-CBD (chitin binding domain) tag behind the bacteriocin gene to better purify our peptide.

Figure 1: BioBrick: T7 promoter + RBS + target peptide + intein-CBD.

Bacteriocin

     It is a peptidic toxin produced by bacteria that inhibits the growth of similar or closely related bacterial strains, but does not damage the bacteria themselve by specific immune proteins. It is heat stable and shows its antimicrobial activity in various temperatures and pH levels. Unlike most antibiotics, which are secondary metabolites, bacteriocins are ribosomally synthesized and sensitive to proteases while generally harmless to the human body and surrounding environment.

     Most bacteriocins interact with anionic lipids that are abundantly present in the membranes of gram-positive bacteria. The receptors on the target membrane can determine the specificity of bacteriocins, and the insertion of bacteriocins is driven by proton motive force.

Figure 2: Mechanism of bacteriocin.

Purification

     Intein is a protein segment that is able to excise itself from the larger protein it binds to. Therefore, it is also called “ protein introns”. We utilized intein-CBD tag to purify our peptides.
    Although affinity tags have been widely used to purify recombinant proteins, it must removed by protease in the final purification. In contrast, intein-CBD tag will undergo the cleavage reaction with DTT (1,4-dithiothreitol) or cysteine, which will not cause the structure changes of our short peptide.

Figure 3: Procedure of intein-mediated protein splicing

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PrOtEin
ExprEssiOn

FunctiOnAl
AnAlysis

Curcumin
BiO-sEnsOr

We expressed our proteins by E. coli BL21 Rosetta-gami.

We checked the inhibitory activity of our bio-stimulator, bacteriocins. The analysis included inhibition zone and inhibition ability.

The detection of curcumin can perfect our productivity model.

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