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<div class="logo-font android-slogan" style="color:#388E3C;">Process</div> | <div class="logo-font android-slogan" style="color:#388E3C;">Process</div> | ||
<div class="logo-font android-sub-slogan" style="color:#757575;"> | <div class="logo-font android-sub-slogan" style="color:#757575;"> | ||
− | + | Here is the way we achieve our design<br> | |
<i class="material-icons">expand_more</i> | <i class="material-icons">expand_more</i> | ||
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Revision as of 18:41, 1 November 2017
Tongji iGEM
TongJi iGEM
Process
Here is the way we achieve our design
expand_more
expand_more
Backbone
![Plasmid Backbone](https://static.igem.org/mediawiki/2017/5/54/2017tongji_wiki_image_process_1.jpeg)
Clone pleP, Gal4, Gal80 and TH
Gal4 (2646bp) integrated with UAS-Gal4 system, and pleP (452bp) and Gal80ts (1311bp) are from the genome of Drosophila melanogaster integrated with a mutant Gal80ts. Because of the repetitive sequence on the TH, it is difficult to clone so we order the synthetic pUAST (double digestion with EcoRI and XbaI)-TH from GENEWIZ®.
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/9/96/File-File-2017tongji_wiki_image_process_4.png)
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/9/98/File-2017tongji_wiki_image_process_2.png)
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/9/96/File-2017tongji_wiki_image_process_3.png)
Digest pUAST-3xHA
For constructing the plasmid pleP-Gal4 and pleP-Gal80ts, avoiding the influence of UAS on the backbone we should remove it. We remove UAS by BamHI digestion.
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/3/3c/2017tongji_wiki_image_process_5.png)
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/e/e4/-2017tongji_wiki_image_process_6.png)
A) pUAST-3xHA
B) pUAST-3xHA BamHI (for 6)
B) pUAST-3xHA BamHI (for 6)
Assembly
To assembly the digested backbone and fragment. We take two ways: to insert a new MCS into the backbone (done), then use a method of double digestion to insert two fragments one by one (failed), or to single digest, then use seamless cloning to insert two fragment together (success).
A) To insert a new MCS into the backbone (done), then use a method of double digestion to insert two fragments one by one (failed)
1. Transform backbone with a new MCS
2. BglII digestion to insert pleP (done)
3. pUAST-new MCS-pleP digestion (done) to insert Gal4 and Gal80ts (failed)
B) To single digest, then use seamless cloning to insert two fragment together (success)
1. BamHI digestion (2 done)
2. Seamless cloning to insert two fragment together (done)
We have done the enzyme digestion to verify them
A) To insert a new MCS into the backbone (done), then use a method of double digestion to insert two fragments one by one (failed)
1. Transform backbone with a new MCS
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/1/1f/2017tongji_wiki_image_process_7.png)
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/0/03/2017tongji_wiki_image_process_8.png)
2. BglII digestion to insert pleP (done)
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/5/5d/2017tongji_wiki_image_process_9.png)
Digestion
A) pUAST-oligo BglII
B) pUAST-oligo
A) pUAST-oligo BglII
B) pUAST-oligo
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/4/44/2017tongji_wiki_image_process_10.png)
pUAST-oligo-pleP
3. pUAST-new MCS-pleP digestion (done) to insert Gal4 and Gal80ts (failed)
![NO DESCRIPTION](https://static.igem.org/mediawiki/2017/e/e9/2017tongji_wiki_image_process_11.png)
Digestion
A) pUAST-new MCS -pleP
B) pUAST-new MCS -pleP NotI
C) pUAST-new MCS -pleP HpaI
D) pUAST-new MCS -pleP NotI&HpaI (For 11)
A) pUAST-new MCS -pleP
B) pUAST-new MCS -pleP NotI
C) pUAST-new MCS -pleP HpaI
D) pUAST-new MCS -pleP NotI&HpaI (For 11)
B) To single digest, then use seamless cloning to insert two fragment together (success)
1. BamHI digestion (2 done)
2. Seamless cloning to insert two fragment together (done)
We have done the enzyme digestion to verify them
![1% gel electrophoresis of plasmid](https://static.igem.org/mediawiki/2017/e/e3/2017tongji_wiki_image_process_12.png)
pUAST-pleP-Gal4
A) pUAST-pleP-Gal4
B) pUAST-pleP-Gal4 PstI
A) pUAST-pleP-Gal4
B) pUAST-pleP-Gal4 PstI
![1% gel electrophoresis of plasmid](https://static.igem.org/mediawiki/2017/b/b8/2017tongji_wiki_image_process_13.png)
pUAST-pleP-Gal80ts
A) pUAST-pleP-Gal80ts
B) pUAST-pleP-Gal80ts KpnI
A) pUAST-pleP-Gal80ts
B) pUAST-pleP-Gal80ts KpnI
![1% gel electrophoresis of plasmid](https://static.igem.org/mediawiki/2017/0/0a/2017tongji_wiki_image_process_14.png)
pUAST-UAS-TH
A) pUAST-UAS-TH
B) pUAST-UAS-TH BglII
A) pUAST-UAS-TH
B) pUAST-UAS-TH BglII