Difference between revisions of "Team:CCU Taiwan/Results"

 
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<body>
 
<body>
 
   <header>
 
   <header>
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         <nav class="navbar navbar-default navbar-inverse navbar-fixed-top">
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         <nav>
 
             <div class="container">
 
             <div class="container">
 
                 <ul class="front">
 
                 <ul class="front">
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'https://2018.igem.org/Team:CCU_Taiwan';"> <img src="https://static.igem.org/mediawiki/2018/0/08/T--CCU_Taiwan--home_button.png"></img></div>
 
'https://2018.igem.org/Team:CCU_Taiwan';"> <img src="https://static.igem.org/mediawiki/2018/0/08/T--CCU_Taiwan--home_button.png"></img></div>
  
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                     <li class="title" style="cursor:pointer;" id="Home"><img class="img_title" src="https://static.igem.org/mediawiki/2018/2/24/T--CCU_Taiwan--aboutus.png"></img><a>About Us</a><hr>
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                     <li class="title" style="cursor:pointer;" id="Home"><img class="img_title" src="https://static.igem.org/mediawiki/2018/2/24/T--CCU_Taiwan--aboutus.png"></img><a>About Us</a>
 
                         <ul class="sub" id="sub_home" style="cursor:default;">
 
                         <ul class="sub" id="sub_home" style="cursor:default;">
 
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Team"><li class="list" id="home1">Team</li></a>
 
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Team"><li class="list" id="home1">Team</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Attributions"><li class="list" id="home2">Attributions</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Attributions"><li class="list" id="home2">Attributions</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Medal"><li class="list" id="home2">Medals</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Medal"><li class="list" id="home3">Medals</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Judge"><li class="list" id="home3">For Judges</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Judge"><li class="list" id="home4">For Judges</li></a>
 +
<a href="https://2018.igem.org/Team:CCU_Taiwan/Achievements"><li class="list" id="home5">Achievements</li></a>
 
                         </ul>
 
                         </ul>
 
                     </li>
 
                     </li>
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                     <li class="title" style="cursor:pointer;" id="Project"><img class="img_title" src="https://static.igem.org/mediawiki/2018/6/6f/T--CCU_Taiwan--project.png"></img><a>Project</a><hr>
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                     <li class="title" style="cursor:pointer;" id="Project"><img class="img_title" src="https://static.igem.org/mediawiki/2018/6/6f/T--CCU_Taiwan--project.png"></img><a>Project</a>
 
<ul class="sub" id="sub_project" style="cursor:default;">
 
<ul class="sub" id="sub_project" style="cursor:default;">
 
    <a href="https://2018.igem.org/Team:CCU_Taiwan/Description"><li class="list" id="project1">Description</li></a>
 
    <a href="https://2018.igem.org/Team:CCU_Taiwan/Description"><li class="list" id="project1">Description</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Applied_Design"><li class="list" id="project2">Design</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Applied_Design"><li class="list" id="project2">Design</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Results"><li class="list" id="project3">Results</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Results"><li class="list" id="project3">Results</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Demonstrate"><li class="list" id="project6">Demonstration</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Demonstrate"><li class="list" id="project4">Demonstration</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/InterLab "><li class="list" id="project5">InterLab</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/InterLab"><li class="list" id="project5">InterLab</li></a>
 
</ul>
 
</ul>
 
</li>
 
</li>
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                     <li class="title" style="cursor:pointer;" id="Parts"><img class="img_title" src="https://static.igem.org/mediawiki/2018/1/17/T--CCU_Taiwan--part.png"></img><a>Parts</a><hr>
+
                     <li class="title" style="cursor:pointer;" id="Parts"><img class="img_title" src="https://static.igem.org/mediawiki/2018/1/17/T--CCU_Taiwan--part.png"></img><a>Parts</a>
 
                         <ul class="sub" id="sub_parts" style="cursor:default;">
 
                         <ul class="sub" id="sub_parts" style="cursor:default;">
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    <a href="https://2018.igem.org/Team:CCU_Taiwan/Parts"><li class="list" id="parts1">Overview</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Parts"><li class="list" id="parts1">Overview</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Basic_Part"><li class="list" id="parts2">Basic Part</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Basic_Part"><li class="list" id="parts1">Basic Part</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Composite_Part"><li class="list" id="parts3">Composite Part</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Composite_Part"><li class="list" id="parts2">Composite Part</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Improve"><li class="list" id="parts4">Improved Part</li></a>
+
 
                         </ul>
 
                         </ul>
 
                     </li>
 
                     </li>
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                     <li class="title" style="cursor:pointer;" id="Modeling"><img class="img_title" src="https://static.igem.org/mediawiki/2018/0/09/T--CCU_Taiwan--model.png"></img><a>Modeling</a><hr>
+
                     <li class="title" style="cursor:pointer;" id="Modeling"><img class="img_title" src="https://static.igem.org/mediawiki/2018/0/09/T--CCU_Taiwan--model.png"></img><a>Modeling</a>
 
                         <ul class="sub" id="sub_modeling" style="cursor:default;">
 
                         <ul class="sub" id="sub_modeling" style="cursor:default;">
 
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Model"><li class="list" id="model1">Overview</li></a>
 
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Model"><li class="list" id="model1">Overview</li></a>
−
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Binding"><li class="list" id="model2">Binding</li></a>
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                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Binding"><li class="list" id="model2">Binding Model</li></a>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/Polymer"><li class="list" id="model4">Polymer</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Polymer"><li class="list" id="model3">Polymer Model</li></a>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/Degradation"><li class="list" id="model3">Degradation</li></a>
+
 
                         </ul>
 
                         </ul>
 
                     </li>
 
                     </li>
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                     <li class="title" style="cursor:pointer;" id="Drylab"><img class="img_title" src="https://static.igem.org/mediawiki/2018/f/fc/T--CCU_Taiwan--Dry_lab.png"></img><a>Product</a><hr>
+
                     <li class="title" style="cursor:pointer;" id="Drylab"><img class="img_title" src="https://static.igem.org/mediawiki/2018/f/fc/T--CCU_Taiwan--Dry_lab.png"></img><a>Product</a>
 
                         <ul class="sub" id="sub_drylab" style="cursor:default;">
 
                         <ul class="sub" id="sub_drylab" style="cursor:default;">
 
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Our_Plan"><li class="list" id="drylab1">Analysis</li></a>
 
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Our_Plan"><li class="list" id="drylab1">Analysis</li></a>
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                         </ul>
 
                         </ul>
 
                     </li>
 
                     </li>
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    <li class="title" style="cursor:pointer;" id="Human_Practice"><img class="img_title" src="https://static.igem.org/mediawiki/2018/9/96/T--CCU_Taiwan--humanpractice.png"></img><a>HP</a><hr>
+
    <li class="title" style="cursor:pointer;" id="Human_Practice"><img class="img_title" src="https://static.igem.org/mediawiki/2018/9/96/T--CCU_Taiwan--humanpractice.png"></img><a>HP</a>
 
                         <ul class="sub" id="sub_human_practice" style="cursor:default;">
 
                         <ul class="sub" id="sub_human_practice" style="cursor:default;">
−
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Human_Practices"><li class="list" id="human_practice1">Overview</li></a>
+
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Human_Practices"><li class="list" id="human_practice1">Human Practice</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Public_Engagement"><li class="list" id="human_practice2">Public Engagement</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Public_Engagement"><li class="list" id="human_practice2">Public Engagement</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Entrepreneurship"><li class="list" id="human_practice3">Entrepreneurship</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Entrepreneurship"><li class="list" id="human_practice3">Entrepreneurship</li></a>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/Intergrate"><li class="list" id="human_practice4">Intergrated HP</li></a>
+
<a href="https://2018.igem.org/Team:CCU_Taiwan/engaging_experts"><li class="list" id="human_practice4">Engaging Experts</li></a>
 +
<a href="https://2018.igem.org/Team:CCU_Taiwan/Integrate"><li class="list" id="human_practice5">Integrated HP</li></a>
 
                         </ul>
 
                         </ul>
 
                     </li>
 
                     </li>
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                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Notebook"><li class="list" id="notebook1">Overview</li></a>
 
                             <a href="https://2018.igem.org/Team:CCU_Taiwan/Notebook"><li class="list" id="notebook1">Overview</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Collaborations"><li class="list" id="notebook2">Collaborations</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Collaborations"><li class="list" id="notebook2">Collaborations</li></a>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/Protocols"><li class="list" id="notebook2">Protocols</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Protocols"><li class="list" id="notebook3">Protocols</li></a>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/Experiments"><li class="list" id="notebook3">Experiments</li></a>
+
<a href="https://2018.igem.org/Team:CCU_Taiwan/Experiments"><li class="list" id="notebook4">Experiments</li></a>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/Materials"><li class="list" id="notebook4">Materials</li></a>
+
<a href="https://2018.igem.org/Team:CCU_Taiwan/Materials"><li class="list" id="notebook5">Materials</li></a>
 
                         </ul>
 
                         </ul>
 
                     </li>
 
                     </li>
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     </header>
 
     </header>
 +
<div class="indicator">
 +
 +
<div class="pointerProject" id="2"><a href="#ca1">Project Achievements</a></div>
 +
 +
</div>
  
 
<div class="backgroundProject">
 
<div class="backgroundProject">
−
<div class="photoResult"></div>
+
<div class="photoResult"><h1 class="bigtitle">RESULTS<h1></div>
 
       <div class="content">
 
       <div class="content">
−
 
+
<p class="first" id="ca1">Project Achievements</p>
−
<p class="first">Result</p>
+
<p class="description">
−
<p class="description">&emsp;&emsp;In our project, we have completed a production system for enzymes ---- peroxidase and laccase. With the high expression of p-pastoris, we have successfully achieved stable enzyme production, we will design production line to mass production in the future.<br><br>
+
1. Prove our vectors successfully constructed<br>
−
&emsp;&emsp;With our improved engineering yeast, we can achieve high-efficiency fermentation production. In general, enzymes production requires cell disruption, and our bacteria can produce exocrine enzymes, then our production line will proceed more efficiently. The overall production line design is also referenced to the enterprise, so it will be smoother. In the end, our market estimate is actually designed in accordance with the relevant industries, and there are related paper mills have expectations for our products. We believe that our project can really enter the market in the future.
+
2. Prove three enzymes successfully produced<br>
−
</p>
+
3. Prove enzymes are functional<br>
−
<br>
+
</p>
−
<br>
+
<p class="second">Prove our vectors successfully constructed</p>
−
<br>
+
<p class="description">
−
      </div>
+
&emsp;&emsp;In LIGGREEN synthesis, monolignols react mainly with two enzymes, peroxidase and laccase. We decided to synthesize these enzymes through synthetic biology. In our experiments, we use <I>P. pastoris</I> as our host. Our goal is to transform the insert into <I>P. pastoris</I>. We did westernblot to prove the yeast transform was successful and the enzymes were produced.
 +
</p>
 +
<div id="res1" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/2018/5/57/T--CCU_Taiwan--JOE_results_figure1.jpg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 1：Plasmid digest( EcoRI, AgeI) after miniprep (pGAPZ A-SalI_His4, pGAPZ A-SalI)</p>
 +
                  </div>
 +
        </div>
 +
<div id="res2" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/2018/0/0e/T--CCU_Taiwan--JOE_results_figure2.jpg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 2：Plasmid digest( EcoRI, AgeI) after miniprep (Px16, Px18, Lac1)</p>
 +
                  </div>
 +
        </div>
 +
<br><br>
 +
<div id="res3" class="polaroid">
 +
                  <img src=" https://static.igem.org/mediawiki/2018/6/6b/T--CCU_Taiwan--JOE_results_figure3.jpg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 3：PCR result of E.coli colony (pGAPZ A_Px16, pGAPZ A_Px18, pGAPZ A_Lac1)</p>
 +
                  </div>
 +
        </div>
 +
<br><br>
 +
<p class="description">&emsp;&emsp;According to gel electrophoresis, we have confirm that our cloning is successful.</p>
 +
<p class="second">Prove three enzymes successfully produced</p>
 +
<p class="description">
 +
&emsp;&emsp;We use western blot to analyze our protein expression. To detect Lac1, Px16, Px18, we select mouse anti-FLAG tag, mouse anti-HA tag, mouse anti-V5 tag, respectively as our primary antibody. As our secondary antibody, we use goat anti-mouse HRP. We to detect secondary antibody, we apply standard enhanced chemilumescent (ECL) substrates for detecting horseradish peroxidase (HRP) enzyme activity.
 +
</p>
 +
<p class="description">
 +
Western blot：
 +
</p>
 +
<div id="res4" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/2018/8/87/T--CCU_Taiwan--western_blot_%28lac1_protein_extraction_from_pellet%29.jpeg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 4：Western blot (lac1 protein extraction from pellet) of high copy strain, middle copy strain, high copy strain at 96hr</p>
 +
                  </div>
 +
        </div>
 +
<div id="res5" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/2018/9/90/T--CCU_Taiwan--western_blot_%28prx18_protein_extraction_from_pellet%29.png" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 5：Western blot (px18 protein extraction from pellet) of high copy strain</p>
 +
                  </div>
 +
        </div><br><br><br>
 +
<div id="res6" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/2018/e/e5/T--CCU_Taiwan--western_blot_%28px18_soup%29.png" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 6：Western blot (px18 protein extraction from soup) of high copy strain</p>
 +
                  </div>
 +
        </div>
 +
<div id="res7" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/2018/6/65/T--CCU_Taiwan--western_blot_%28px16_protein_extraction_from_pellet%29.png" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 7：Western blot (px16 protein extraction from pellet) of low copy strain</p>
 +
                  </div>
 +
        </div><br><br><br><br>
 +
<p class="second">Prove enzymes are functional</p>
 +
<p class="description">
 +
&emsp;&emsp;Coniferyl alcohol will react with our enzymes, Px16, Px18 and Lac1. We want to prove that coniferyl alcohol will polymerize into an oligomer. We use UV-visible for measurements. Through UV-visible, we can prove the difference in absorbance wavelength between monomer and oligomer.
 +
</p>
 +
<p class="description">
 +
UV-visible :
 +
</p>
 +
<div id="res8" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/2018/4/48/T--CCU_Taiwan--JOE_results_4-5.jpg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 8：the difference between monomer and monomer + Lac1</p>
 +
                  </div>
 +
        </div><br><br>
 +
<div id="res9" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/2018/f/fb/T--CCU_Taiwan--JOE_results_4-6.jpg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 9：the difference between monomer and monomer + Px16 and Px18 </p>
 +
                  </div>
 +
        </div><br><br>
 +
<div id="res10" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/2018/a/a9/T--CCU_Taiwan--JOE_results_4-7.jpg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 10：the difference between monomer and monomer + Lac1 + Px16 and Px18</p>
 +
                  </div>
 +
        </div><br><br><br><br>
 +
<p class="description">
 +
&emsp;&emsp;According to UV-visible, when the wavelength reaches some specific positions, the oligomer will shift to the left more than the monomer. Therefore, we found that only the peroxidase case did not produce spectral absorption. We have shown that our enzymes can polymerize the monomer into the oligomer.
 +
</p><br><br><br><br><br><br>
 +
    </div>
 
</div>
 
</div>
 
</body>
 
</body>

Latest revision as of 08:46, 1 December 2018

RESULTS

Project Achievements

1. Prove our vectors successfully constructed
2. Prove three enzymes successfully produced
3. Prove enzymes are functional

Prove our vectors successfully constructed

  In LIGGREEN synthesis, monolignols react mainly with two enzymes, peroxidase and laccase. We decided to synthesize these enzymes through synthetic biology. In our experiments, we use P. pastoris as our host. Our goal is to transform the insert into P. pastoris. We did westernblot to prove the yeast transform was successful and the enzymes were produced.

Figure 1：Plasmid digest( EcoRI, AgeI) after miniprep (pGAPZ A-SalI_His4, pGAPZ A-SalI)

Figure 2：Plasmid digest( EcoRI, AgeI) after miniprep (Px16, Px18, Lac1)



Figure 3：PCR result of E.coli colony (pGAPZ A_Px16, pGAPZ A_Px18, pGAPZ A_Lac1)



  According to gel electrophoresis, we have confirm that our cloning is successful.

Prove three enzymes successfully produced

  We use western blot to analyze our protein expression. To detect Lac1, Px16, Px18, we select mouse anti-FLAG tag, mouse anti-HA tag, mouse anti-V5 tag, respectively as our primary antibody. As our secondary antibody, we use goat anti-mouse HRP. We to detect secondary antibody, we apply standard enhanced chemilumescent (ECL) substrates for detecting horseradish peroxidase (HRP) enzyme activity.

Western blot：

Figure 4：Western blot (lac1 protein extraction from pellet) of high copy strain, middle copy strain, high copy strain at 96hr

Figure 5：Western blot (px18 protein extraction from pellet) of high copy strain




Figure 6：Western blot (px18 protein extraction from soup) of high copy strain

Figure 7：Western blot (px16 protein extraction from pellet) of low copy strain





Prove enzymes are functional

  Coniferyl alcohol will react with our enzymes, Px16, Px18 and Lac1. We want to prove that coniferyl alcohol will polymerize into an oligomer. We use UV-visible for measurements. Through UV-visible, we can prove the difference in absorbance wavelength between monomer and oligomer.

UV-visible :

Figure 8：the difference between monomer and monomer + Lac1



Figure 9：the difference between monomer and monomer + Px16 and Px18



Figure 10：the difference between monomer and monomer + Lac1 + Px16 and Px18





  According to UV-visible, when the wavelength reaches some specific positions, the oligomer will shift to the left more than the monomer. Therefore, we found that only the peroxidase case did not produce spectral absorption. We have shown that our enzymes can polymerize the monomer into the oligomer.