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> | ||
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<p class="description">  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">  It will produce the mature laccase 1 without Prepro-alpha-factor, and the molecular weight of enzyme will be 63.65 kilodaltons. | ||
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<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> | ||
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<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">  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">  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. | ||
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<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> | ||
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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