Team:Tsinghua/Parts

From 2014.igem.org

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     <h1>Collaboration</h1>
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     <h1>Parts</h1>
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<p>As one of the 254 teams that have joined the big family of iGEM, we are trying our best to meet new friends and offer help to those who are in need. That is one thing that makes this competition interesting and beautiful, it not all about competing, but also about communicating and collaborating.</p>
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<h3>&nbsp;</h3>
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<h2>Collaboration with Tsinghua-A</h2>
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<h2>2014-PDX-1</h2>
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<p>We have been in close collaboration with another team from our home university, <a href=” https://2014.igem.org/Team:Tsinghua-A”>Tsinghua-A</a>. They are excellent team and are doing amazing work. We feel very happy that we were able to give them hands on their experiment and part submission. It was also quite exciting when we were holding a mini-symposium together for students in our school. We talked about our projects and also tried to present the beauty of synthetic biology to junior students. </p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2014/5/5f/2014_thu_parts1.png"></div>
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<h2>Collaboration with BNU-China</h2>
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<p>PDX-1 is a transcriptional factor that activates insulin, somatostatin, glucokinase, islet amyloid polypeptide and glucose transporter type 2 gene transcription. It’s essential for development of pancreas and maintain of the hormone-producing phenotype of the beta-cell. PDX-1 is used here for glucose-dependent regulation of insulin gene expression.</p>
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<p>Our team has been participating in iGEM for the past five years and much experience has been passed down from one generation to another. As such an experienced team, we are very willing to help out those new teams which probably join the iGEM for the first time this year. This is what we did for the team from Beijing Normal University (<a href=” https://2014.igem.org/Team:BNU-China”>BNU-China</a>). They are a team of rising stars and are willing to take initiative. We are very happy to be able to help. The two teams scheduled several meetings and we were having a wonderful time. </p>
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<h3>PDX-1</h3>
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<p>It is natural for contestants to be curious about what their competitors are doing. The forum organized by NCTU provided a perfect opportunity for every team attended to satisfy their curiosity. During the Forum, we communicated with teams from universities all over China. Posters and presentations were made to introduce every team’s design and progress; questions were raised as well as helpful advices. Every one attended really touched the thrill of iGemer’s happiness, and got home fully load. </p>
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<p>PDX-1 is a transcription factor involved in the expression of insulin and insulin-related genes. Co-transfection of PDX-1 and MafA in mammalian cells induces cell-type conversion to insulin producing beta-cells.</p>
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    <h3>&nbsp;</h3>
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<p>We are very lucky to have the opportunity to join the big family of iGEM and put our ideas into reality, and it is even luckier that we are able to witness the clash of ideas and the genesis of even crazier ideas. Collaboration is what makes this competition complicated but is also what makes it inspiring and long-lasting. </p>
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<h2>2014-insulin</h2>
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<div align="center"><img src="https://static.igem.org/mediawiki/2014/0/07/2014_thu_parts2.png"></div>
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<p>The insulin gene consists three exons, which encode B chain, C peptide and A chain. We modified the protease recognition sites of insulin gene to make sure that the insulin gene product undergoes accurate modification. The signal peptide and C peptide are removed after transcription and translation and two disulfide bonds form between A chain and B chain, undergoing folding to become functional insulin.</p>
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<h3>Insulin (modified protease recognition sites)</h3>
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<p>Encodes the human insulin gene (full length). Two 6 bp sequences were inserted into two separate sites within the insulin gene to allow enzymatic digestion by the protease furin. Originally, proinsulin is cleaved three times with three different enzymes, prohormone convertases (PC1 and PC2), and carboxypeptidase E before being secreted as mature insulin. Since PC1 and PC2 expression is tissue specific, we replaced their recognition sites with that of furin, which is universally expressed, thus allowing this insulin gene to be expressed and processed in any somatic cell type.</p>
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    <h3>&nbsp;</h3>
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<h2>2014-HXT1+CMV</h2>
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<div align="center"><img src="https://static.igem.org/mediawiki/2014/7/77/2014_thu_parts3.png"></div>
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<p>Part HXT1+CMV is used as promoter for target gene under the regulation of glucose, consisting of HXT-1 promoter and CMV promoter. HXT-1 is a low-affinity glucose transporter from Saccharomyces cerevisiae. There is a glucose response element in HXT-1 gene sensitive to glucose in human fibroblasts, which is used here as a glucose-dependent regulator.</p>
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    <h3>&nbsp;</h3>
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<h2>2014-HXT1+CMV+EGFP+ADH1</h2>
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<div align="center"><img src="https://static.igem.org/mediawiki/2014/e/e3/2014_thu_parts4.png"></div>
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<p>HXT-1+CMV+EGFP+ADH1 consists of HXT-1+CMV promoter, EGFP and terminator ADH1. HXT-1+CMV promoter controls EGFP expression under the regulation of glucose. EGFP is used here for testing of HXT1+CMV promoter and will be replaced by insulin gene.</p>
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<h3>HXT1-CMVmini-EGFP-tADH1</h3>
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<p>This is a composite part containing a glucose-sensitive promoter (BBa_K1328000) and a EGFP reporter with a tADH1 transcription terminator.</br>
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S. cerevisiae cells transformed with this part was cultured in glucose-free medium with glycerol as carbon-source for 8 hrs. They were then cultured for an additional hour with varying glucose levels (0, 5, 15, 25 mmol/L). A increasing level of GFP expression was observed with increasing levels of glucose concentration, in both microscopy and flow cytometry observations.
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</p>
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    <h3>&nbsp;</h3>
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Latest revision as of 20:19, 17 October 2014

Team Tsinghua 2014 Return to iGEM 2014

Parts

 

2014-PDX-1

PDX-1 is a transcriptional factor that activates insulin, somatostatin, glucokinase, islet amyloid polypeptide and glucose transporter type 2 gene transcription. It’s essential for development of pancreas and maintain of the hormone-producing phenotype of the beta-cell. PDX-1 is used here for glucose-dependent regulation of insulin gene expression.

PDX-1

PDX-1 is a transcription factor involved in the expression of insulin and insulin-related genes. Co-transfection of PDX-1 and MafA in mammalian cells induces cell-type conversion to insulin producing beta-cells.

 

2014-insulin

The insulin gene consists three exons, which encode B chain, C peptide and A chain. We modified the protease recognition sites of insulin gene to make sure that the insulin gene product undergoes accurate modification. The signal peptide and C peptide are removed after transcription and translation and two disulfide bonds form between A chain and B chain, undergoing folding to become functional insulin.

Insulin (modified protease recognition sites)

Encodes the human insulin gene (full length). Two 6 bp sequences were inserted into two separate sites within the insulin gene to allow enzymatic digestion by the protease furin. Originally, proinsulin is cleaved three times with three different enzymes, prohormone convertases (PC1 and PC2), and carboxypeptidase E before being secreted as mature insulin. Since PC1 and PC2 expression is tissue specific, we replaced their recognition sites with that of furin, which is universally expressed, thus allowing this insulin gene to be expressed and processed in any somatic cell type.

 

2014-HXT1+CMV

Part HXT1+CMV is used as promoter for target gene under the regulation of glucose, consisting of HXT-1 promoter and CMV promoter. HXT-1 is a low-affinity glucose transporter from Saccharomyces cerevisiae. There is a glucose response element in HXT-1 gene sensitive to glucose in human fibroblasts, which is used here as a glucose-dependent regulator.

 

2014-HXT1+CMV+EGFP+ADH1

HXT-1+CMV+EGFP+ADH1 consists of HXT-1+CMV promoter, EGFP and terminator ADH1. HXT-1+CMV promoter controls EGFP expression under the regulation of glucose. EGFP is used here for testing of HXT1+CMV promoter and will be replaced by insulin gene.

HXT1-CMVmini-EGFP-tADH1

This is a composite part containing a glucose-sensitive promoter (BBa_K1328000) and a EGFP reporter with a tADH1 transcription terminator.
S. cerevisiae cells transformed with this part was cultured in glucose-free medium with glycerol as carbon-source for 8 hrs. They were then cultured for an additional hour with varying glucose levels (0, 5, 15, 25 mmol/L). A increasing level of GFP expression was observed with increasing levels of glucose concentration, in both microscopy and flow cytometry observations.

 

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