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

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                     <p>Figure 1: The schematic structure of gene design- Lac1.</p>
 
                     <p>Figure 1: The schematic structure of gene design- Lac1.</p>
 
                   </div>
 
                   </div>
                 </div>
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                 </div><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 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>
+
</p><br>
 
        <div id="Composite1-2" class="polaroid">
 
        <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%">
 
                   <img src="https://static.igem.org/mediawiki/parts/2/2f/T--CCU_Taiwan--_lac1_western_blot.jpeg" style="width:100%">
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                   </div>
 
                   </div>
 
                 </div>
 
                 </div>
 
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<br><br><br>
  
 
<p class="second"><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>
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                 </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 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>
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</p><br>
 
        <div id="Composite2-2" class="polaroid">
 
        <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%">
 
                   <img src="https://static.igem.org/mediawiki/parts/6/62/T--CCU_Taiwan--_px16_western_blot-p.png" style="width:100%">
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                   </div>
 
                   </div>
 
                 </div>
 
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<br>
 +
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        <div id="Composite3-3" class="polaroid">
 
        <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%">
 
                   <img src="https://static.igem.org/mediawiki/parts/3/37/T--CCU_Taiwan--_px18_western_blot-p.png" style="width:100%">
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               <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">

Revision as of 22:42, 17 October 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