Difference between revisions of "Team:CCU Taiwan/Composite Part"

 
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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>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/Intergrate"><li class="list" id="human_practice5">Intergrated HP</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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     </header>
 
     </header>
 +
<div class="indicator">
  
 +
<div class="pointerProject" id="2"><a href="#ca1">laccase 1</a></div>
 +
<div class="pointerProject" id="3"><a href="#ca2">peroxidase 18</a></div>
 +
<div class="pointerProject" id="4"><a href="#ca3">peroxidase 16</a></div>
 +
</div>
 
 
 
<div class="backgroundParts">
 
<div class="backgroundParts">
 
<div class="photoComposite"><h1 class="bigtitle">COMPOSITE PART<h1></div>
 
<div class="photoComposite"><h1 class="bigtitle">COMPOSITE PART<h1></div>
 
<div class="content">
 
<div class="content">
−
<p class="second"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2809011">BBa_K2809011</a><br>
+
<p class="second" id="ca1"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2809011">BBa_K2809011</a><br>
 
laccase 1
 
laccase 1
 
</p>
 
</p>
−
<p class="description">We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809010.<br>
+
<p class="description">&emsp;&emsp;We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809010.<br>
−
   This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of laccase gene sequence, HA tag for Western Blot protein detection and 6xHis-tag for protein purification.  
+
   &emsp;&emsp;This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of laccase gene sequence, HA tag for Western Blot protein detection and 6xHis-tag for protein purification.  
−
</p>
+
</p><br>
  
 
        <div id="Composite1" class="polaroid">
 
        <div id="Composite1" class="polaroid">
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                   <div class="container">
 
                   <div class="container">
 
                     <p>Figure 1: The schematic structure of gene design- Lac1.</p>
 
                     <p>Figure 1: The schematic structure of gene design- Lac1.</p>
 +
                  </div>
 +
                </div><br><br>
 +
<p class="description">&emsp;&emsp;It will produce the mature laccase 1 without Prepro-alpha-factor, and the molecular weight of enzyme will be 63.65 kilodaltons.
 +
</p><br>
 +
        <div id="Composite1-2" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/parts/2/2f/T--CCU_Taiwan--_lac1_western_blot.jpeg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 2: The result of western blot— laccase 1.</p>
 
                   </div>
 
                   </div>
 
                 </div>
 
                 </div>
 +
<br><br><br>
  
−
 
+
<p class="second" id="ca2"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2809021">BBa_K2809021</a><br>
−
 
+
−
<p class="second"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2809021">BBa_K2809021</a><br>
+
 
peroxidase 18
 
peroxidase 18
 
</p>
 
</p>
−
<p class="description"> We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809020.<br>
+
<p class="description">&emsp;&emsp;We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809020.<br>
 
   This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of peroxidase 18 gene sequence, V5-tag for Western Blot protein detection and 6xHis-tag for protein purification.  
 
   This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of peroxidase 18 gene sequence, V5-tag for Western Blot protein detection and 6xHis-tag for protein purification.  
 
   
 
   
−
</p>
+
</p><br><br>
 
        <div id="Composite2" class="polaroid">
 
        <div id="Composite2" class="polaroid">
 
                   <img src="https://static.igem.org/mediawiki/parts/9/9e/T--CCU_Taiwan--114.png" style="width:100%">
 
                   <img src="https://static.igem.org/mediawiki/parts/9/9e/T--CCU_Taiwan--114.png" style="width:100%">
 
                   <div class="container">
 
                   <div class="container">
−
                     <p>Figure 2: The schematic structure of gene design- Px18.</p>
+
                     <p>Figure 3: The schematic structure of gene design- Px18.</p>
 
                   </div>
 
                   </div>
 
                 </div><br><br>
 
                 </div><br><br>
−
 
+
<p class="description">&emsp;&emsp;It will produce the mature peroxidase 16 without Prepro-alpha-factor, and the molecular weight of enzyme should be 35.29 kilodaltons. However, because of protein N-linked glycosylation, the molecular weight of enzyme will be about 37~45 kilodaltons according to the Western blot result.
−
 
+
</p><br>
 +
        <div id="Composite2-2" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/parts/6/62/T--CCU_Taiwan--_px16_western_blot-p.png" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 4: The result of Western blot— peroxidase 16.</p>
 +
                  </div>
 +
                </div><br><br
 
<br>
 
<br>
−
<p class="second"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2809031">BBa_K2809031</a><br>
+
<p class="second" id="ca3"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2809031">BBa_K2809031</a><br>
 
peroxidase 16
 
peroxidase 16
 
</p>
 
</p>
−
<p class="description"> We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809030.<br>
+
<p class="description">&emsp;&emsp;We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809030.<br>
 
   This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of peroxidase 16 gene sequence, FLAG-tag for Western Blot protein detection and 6xHis-tag for protein purification.  
 
   This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of peroxidase 16 gene sequence, FLAG-tag for Western Blot protein detection and 6xHis-tag for protein purification.  
  
−
</p><br>         <div id="Composite3" class="polaroid">
+
</p><br><br>         <div id="Composite3" class="polaroid">
 
                   <img src="https://static.igem.org/mediawiki/parts/1/1e/T--CCU_Taiwan--117.png" style="width:100%">
 
                   <img src="https://static.igem.org/mediawiki/parts/1/1e/T--CCU_Taiwan--117.png" style="width:100%">
 
                   <div class="container">
 
                   <div class="container">
−
                     <p>Figure 3: The schematic structure of gene design- Px16.</p>
+
                     <p>Figure 5: The schematic structure of gene design- Px16.</p>
 
                   </div>
 
                   </div>
 
                 </div><br><br>
 
                 </div><br><br>
 +
<p class="description">&emsp;&emsp;We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809030.<br>
 +
  This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of peroxidase 16 gene sequence, FLAG-tag for Western Blot protein detection and 6xHis-tag for protein purification.
 +
 +
</p><br><br>
 +
        <div id="Composite3-2" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/parts/4/4b/T--CCU_Taiwan--_px18_western_blot-s.png" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 6: The result of Western blot— peroxidase 18(soup).</p>
 +
                  </div>
 +
                </div>
 +
<br>
 +
<br>
 +
        <div id="Composite3-3" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/parts/3/37/T--CCU_Taiwan--_px18_western_blot-p.png" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 7: The result of Western blot— peroxidase 18(protein extraction from pellet).</p>
 +
                  </div>
 +
                </div>
 +
 +
 +
 +
  
 +
<br><br><br>
 
               <p class="second">Part construction in pGAPZ A:</p><br>
 
               <p class="second">Part construction in pGAPZ A:</p><br>
 
<div class="row">
 
<div class="row">

Latest revision as of 08:47, 1 December 2018

COMPOSITE PART

BBa_K2809011
laccase 1

  We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809010.
  This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of laccase gene sequence, HA tag for Western Blot protein detection and 6xHis-tag for protein purification.


Figure 1: The schematic structure of gene design- Lac1.



  It will produce the mature laccase 1 without Prepro-alpha-factor, and the molecular weight of enzyme will be 63.65 kilodaltons.


Figure 2: The result of western blot— laccase 1.




BBa_K2809021
peroxidase 18

  We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809020.
This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of peroxidase 18 gene sequence, V5-tag for Western Blot protein detection and 6xHis-tag for protein purification.



Figure 3: The schematic structure of gene design- Px18.



  It will produce the mature peroxidase 16 without Prepro-alpha-factor, and the molecular weight of enzyme should be 35.29 kilodaltons. However, because of protein N-linked glycosylation, the molecular weight of enzyme will be about 37~45 kilodaltons according to the Western blot result.


Figure 4: The result of Western blot— peroxidase 16.



BBa_K2809031
peroxidase 16

  We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809030.
This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of peroxidase 16 gene sequence, FLAG-tag for Western Blot protein detection and 6xHis-tag for protein purification.



Figure 5: The schematic structure of gene design- Px16.



  We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809030.
This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of peroxidase 16 gene sequence, FLAG-tag for Western Blot protein detection and 6xHis-tag for protein purification.



Figure 6: The result of Western blot— peroxidase 18(soup).



Figure 7: The result of Western blot— peroxidase 18(protein extraction from pellet).




Part construction in pGAPZ A:


Figure 4: PCR result.
Well1: Marker/ Well2, 3, 4: Lac1 Colony 1, 2, 3/ Well5, 6, 7: Px16 Colony 1, 2, 3/ Well8, 9, 10: Px18 Colony 1, 2, 3/ Well11: pGAPZ A Colony
Insert long after PCR — pGAPZ A_Px16 :1377 bp/ pGAPZ A_Px18 :1359 bp/ pGAPZ A_Lac1 :2124bp

Figure 5: Miniprep plasmid digest by EcoRI, PstI.
Well 1: Marker/ Well2, 3, 4: Plasmid of Lac1 colony1, 2, 3 after EcoRI, AgeI digest/ Well5, 6, 7: Plasmid of Px16 colony1, 2, 3 after EcoRI, AgeI digest/ Well8, 9, 10: Plasmid of Px18 colony1, 2, 3 after EcoRI, AgeI digest
Vector long after digest — pGAPZ A: 2884bp
Insert long after digest — Px16: 1295bp/ Px18: 1277bp/ Lac1: 2042bp