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

 
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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>
  
−
                     <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>
+
                     <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>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/Medal"><li class="list" id="home2">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="home3">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>
−
                     <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>
+
                     <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>
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<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>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Demonstrate"><li class="list" id="project4">Demonstration</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Demonstrate"><li class="list" id="project4">Demonstration</li></a>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/InterLab "><li class="list" id="project5">InterLab</li></a>
+
<a href="https://2018.igem.org/Team:CCU_Taiwan/InterLab"><li class="list" id="project5">InterLab</li></a>
 
</ul>
 
</ul>
 
</li>
 
</li>
−
                     <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;">
 +
<a href="https://2018.igem.org/Team:CCU_Taiwan/Parts"><li class="list" id="parts1">Overview</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Basic_Part"><li class="list" id="parts1">Basic Part</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Basic_Part"><li class="list" id="parts1">Basic Part</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Composite_Part"><li class="list" id="parts2">Composite Part</li></a>
 
<a href="https://2018.igem.org/Team:CCU_Taiwan/Composite_Part"><li class="list" id="parts2">Composite Part</li></a>
−
<a href="https://2018.igem.org/Team:CCU_Taiwan/Improve"><li class="list" id="parts3">Improved Part</li></a>
 
 
                         </ul>
 
                         </ul>
 
                     </li>
 
                     </li>
−
                     <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>
+
                             <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="model3">Polymer</li></a>
+
<a href="https://2018.igem.org/Team:CCU_Taiwan/Polymer"><li class="list" id="model3">Polymer Model</li></a>
 
                         </ul>
 
                         </ul>
 
                     </li>
 
                     </li>
−
                     <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>
Line 101: Line 125:
 
                         </ul>
 
                         </ul>
 
                     </li>
 
                     </li>
−
    <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/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>
Line 124: Line 148:
  
 
     </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="photoBasicPart"></div>
+
<div class="photoComposite"><h1 class="bigtitle">COMPOSITE PART<h1></div>
 
<div class="content">
 
<div class="content">
−
<p class="first">Composite Parts</p>
+
<p class="second" id="ca1"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2809011">BBa_K2809011</a><br>
−
<p class="second">BBa_K2809001 <br>
+
−
Kozak sequence (Pichia pastoris)
+
−
</p>
+
−
<p class="description">The Kozak sequence initiates the translation in Pichia pastoris. This Sequence is copied directly from pCore1 in reference .The start codon is not included.</p>
+
−
+
−
<p class="second">BBa_K2809002<br>
+
−
Prepro-alpha-factor (codon modified for p.pastoris)
+
−
</p>
+
−
<p class="description">Prepro-alpha-factor is the secretion signal peptide from the yeast α-mating factor. This signal peptide directs secreted expression of the recombinant protein, therefore, it allows the exportation of it. Once the recombinant protein is in the endoplasmic reticulum(ER) lumen, this secretion signal will be cleaved.</p>
+
−
+
−
<p class="second">BBa_K2809010<br>
+
−
Mature Lac1 (laccase)
+
−
</p>
+
−
<p class="description">This is the codon-optimized version of laccase derived from Pinus taeda(Loblolly pine). Laccase is involved in monolignol dehydrogenation, using oxygen to oxidize their metal centers to enable catalytical phenol oxidation.It is able to produce dimers and oligomers of coniferyl alcohol, which is involved in the early stage of lignification.</p>
+
−
<p class="description">The signal peptide have removed, so the sequence do NOT contain start codon. Furthermore, it includes HA-tag for Western Blot protein detection and 6xHis-tag for protein purification before stop codon.</p>
+
−
+
−
<p class="second">BBa_K2809020<br>
+
−
Mature Px18 (peroxidase)
+
−
</p>
+
−
<p class="second">BBa_K2809030<br>
+
−
Mature Px16 (peroxidase)
+
−
</p>
+
−
<p class="description">Those are the codon-optimized version of peroxidase derived from Picea abies(Norway spruce). Peroxidases (Px16 and Px18) are involved in monolignol dehydrogenation, using hydrogen peroxide as a substrate. They are able to form polymers from oligomers or dimers, which contribute to lignin polymerization.</p>
+
−
<p class="description">The signal peptide have removed, so the sequence do NOT contain start codon.Moreover, it includes V5-tag for Western Blot protein detection and  6xHis-tag for protein purification before stop codon.</p>
+
−
<p class="description">The signal peptide have removed, so the sequence do NOT contain start codon.Furthermore, it includes FLAG-tag for Western Blot protein detection and 6xHis-tag for protein purification before stop codon.</p>
+
−
<p class="description"><br>Best New Composite parts: </p>
+
−
<p class="second">BBa_K2809011<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>
−
<p class="second">BBa_K2809021<br>
+
 
 +
        <div id="Composite1" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/parts/5/59/T--CCU_Taiwan--111.png" style="width:100%">
 +
                  <div class="container">
 +
                    <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>
 +
<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>
 
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>
−
<p class="second">BBa_K2809031<br>
+
        <div id="Composite2" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/parts/9/9e/T--CCU_Taiwan--114.png" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 3: The schematic structure of gene design- Px18.</p>
 +
                  </div>
 +
                </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>
 +
<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><br>         <div id="Composite3" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/parts/1/1e/T--CCU_Taiwan--117.png" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 5: The schematic structure of gene design- Px16.</p>
 +
                  </div>
 +
                </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>
 +
<div class="row">
 +
        <div id="Composite4" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/parts/f/f2/T--CCU_Taiwan--_pGAPZ_A_Lac1%2C_pGAPZ_A_Px16%2C_pGAPZ_A_Px18_Colony_PCR.jpeg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 4: PCR result.<br> 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
 +
<br>
 +
Insert long after PCR — pGAPZ A_Px16 :1377 bp/ pGAPZ A_Px18 :1359 bp/ pGAPZ A_Lac1 :2124bp
 
</p>
 
</p>
 +
                  </div>
 +
                </div>
 +
        <div id="Composite5" class="polaroid">
 +
                  <img src="https://static.igem.org/mediawiki/parts/a/ae/T--CCU_Taiwan--_pSB1C3_Lac1%2C_pSB1C3_Px16%2C_pSB1C3_Px18_Miniprep_and_digest_with_EcoRI%2CAgeI.jpeg" style="width:100%">
 +
                  <div class="container">
 +
                    <p>Figure 5: Miniprep plasmid digest by EcoRI, PstI.<br>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
 +
<br>
 +
Vector long after digest — pGAPZ A: 2884bp
 +
<br>
 +
Insert long after digest — Px16: 1295bp/ Px18: 1277bp/ Lac1: 2042bp
 +
</p>
 +
                  </div>
 +
                </div>
 +
</div>
 +
<br>
 +
<br>
 +
<br>
 +
 
</div>
 
</div>
 
</div>
 
</div>

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