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

 
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Medal"><li class="list" id="home3">Medals</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Medal"><li class="list" id="home3">Medals</li></a>
 
<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/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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<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/engaging_experts"><li class="list" id="human_practice4">Engaging Experts</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/engaging_experts"><li class="list" id="human_practice4">Engaging Experts</li></a>
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<a href="https://2018.igem.org/Team:CCU_Taiwan/Intergrate"><li class="list" id="human_practice5">Intergrated HP</li></a>
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<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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     </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"><h1 class="bigtitle">RESULTS<h1></div>
 
<div class="photoResult"><h1 class="bigtitle">RESULTS<h1></div>
 
       <div class="content">
 
       <div class="content">
−
<p class="first">PROJECT ACHIEVEMENTS</p>
+
<p class="first" id="ca1">Project Achievements</p>
 
<p class="description">
 
<p class="description">
−
1. Prove our vectors successfully be constructed<br>
+
1. Prove our vectors successfully constructed<br>
−
2. Prove three enzymes successfully be produced<br>
+
2. Prove three enzymes successfully produced<br>
 
3. Prove enzymes are functional<br>
 
3. Prove enzymes are functional<br>
−
4. Establishment of material production line<br>
 
 
</p>
 
</p>
−
<p class="second">Prove our vectors successfully be constructed</p>
+
<p class="second">Prove our vectors successfully constructed</p>
 
<p class="description">
 
<p class="description">
−
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 strain. 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.
+
&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>
 
</p>
 
<div id="res1" class="polaroid">
 
<div id="res1" class="polaroid">
−
                   <img src="https://static.igem.org/mediawiki/2018/5/57/T--CCU_Taiwan--JOE_results_figure1.jpg" style="width:50%">
+
                   <img src="https://static.igem.org/mediawiki/2018/5/57/T--CCU_Taiwan--JOE_results_figure1.jpg" style="width:100%">
 
                   <div class="container">
 
                   <div class="container">
 
                     <p>Figure 1：Plasmid digest( EcoRI, AgeI) after miniprep (pGAPZ A-SalI_His4, pGAPZ A-SalI)</p>
 
                     <p>Figure 1：Plasmid digest( EcoRI, AgeI) after miniprep (pGAPZ A-SalI_His4, pGAPZ A-SalI)</p>
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         </div>
 
         </div>
 
<div id="res2" class="polaroid">
 
<div id="res2" class="polaroid">
−
                   <img src="https://static.igem.org/mediawiki/2018/0/0e/T--CCU_Taiwan--JOE_results_figure2.jpg" style="width:50%">
+
                   <img src="https://static.igem.org/mediawiki/2018/0/0e/T--CCU_Taiwan--JOE_results_figure2.jpg" style="width:100%">
 
                   <div class="container">
 
                   <div class="container">
 
                     <p>Figure 2：Plasmid digest( EcoRI, AgeI) after miniprep (Px16, Px18, Lac1)</p>
 
                     <p>Figure 2：Plasmid digest( EcoRI, AgeI) after miniprep (Px16, Px18, Lac1)</p>
 
                   </div>
 
                   </div>
 
         </div>
 
         </div>
 +
<br><br>
 
<div id="res3" class="polaroid">
 
<div id="res3" class="polaroid">
−
                   <img src=" https://static.igem.org/mediawiki/2018/6/6b/T--CCU_Taiwan--JOE_results_figure3.jpg" style="width:50%">
+
                   <img src=" https://static.igem.org/mediawiki/2018/6/6b/T--CCU_Taiwan--JOE_results_figure3.jpg" style="width:100%">
 
                   <div class="container">
 
                   <div class="container">
 
                     <p>Figure 3：PCR result of E.coli colony (pGAPZ A_Px16, pGAPZ A_Px18, pGAPZ A_Lac1)</p>
 
                     <p>Figure 3：PCR result of E.coli colony (pGAPZ A_Px16, pGAPZ A_Px18, pGAPZ A_Lac1)</p>
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         </div>
 
         </div>
 
<br><br>
 
<br><br>
−
<p class="description">According to gel electrophoresis, we have confirm that our cloning is successful.</p>
+
<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 be produced</p>
+
<p class="second">Prove three enzymes successfully produced</p>
 
<p class="description">
 
<p class="description">
−
This is our original expectation. We want to produce the required enzymes, Px16, Px18 and Lac1, through <I>P. pastoris</I>. We have completed the prove of our part. Then, we have completed the improvement of engineering yeast.
+
&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">
+
−
This is the schematic structure of our gene design. you can see, GAP promoter ,which is constitutive type of promoter, then Kozak sequence, alpha factor ,which is secretion signal for directing secreted expression of the recombinant protein, After that, is our target gene, HA Tag for western blot protein detection, and His tag for protein purification Lastly, AOXI terminator is common one in <I>P. pastoris</I>.<br>
+
−
Up to now, you may already found out we put only one target gene in this backbone
+
−
And your right it is important part of our gene design that is our target genes are separated into three different inserts rather than put it in one.
+
 
</p>
 
</p>
 
<p class="description">
 
<p class="description">
 
Western blot：
 
Western blot：
 
</p>
 
</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="second">Prove enzymes are functional</p>
 
<p class="description">
 
<p class="description">
−
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.
+
&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>
 
<p class="description">
 
<p class="description">
 
UV-visible :
 
UV-visible :
 
</p>
 
</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">
 
<p class="description">
−
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.
+
&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>
+
</p><br><br><br><br><br><br>
 
     </div>
 
     </div>
 
</div>
 
</div>

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.