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| − | <p>During our project we had a great deal of work with different types of promoters - constitutive and indusible. During the planning of the project and the accumulation of knowledge about the various parts, we discovered that much information is lacking about many of these parameters, and the information that exists exists scattered among many places and is not arranged in one place. We chose two modulators - TDH3 [<a href="http://parts.igem.org/Part:BBa_K124002" target="_blank">BBa_K124002</a>] of team iGEM08_Brown and PGK1 [<a href="http://parts.igem.org/Part:BBa_K2110012" target="_blank">BBa_K2110012</a>] of team iGEM16_Tianjin, two strong constitutive permutative promoters, and improved their descriptions and information about them, referring to the main sources we used. We hope that groups coming after us will have faster and more convenient access to information when they come to choose the parameters they want to use.</p> | + | <p>During our project we had a great deal of work with different types of promoters - constitutive and inducible. During the planning of the project and the accumulation of knowledge about the various parts, we discovered that much information is lacking about many of these parameters, and the information that exists is scattered and is not arranged in one place. We chose two modulators - TDH3 [<a href="http://parts.igem.org/Part:BBa_K124002" target="_blank">BBa_K124002</a>] of team iGEM08_Brown and PGK1 [<a href="http://parts.igem.org/Part:BBa_K2110012" target="_blank">BBa_K2110012</a>] of team iGEM16_Tianjin, two strong constitutive permutative promoters, and improved their descriptions and the information about them, referring to the main sources we used. We hope that groups coming after us will have faster and more convenient access to information when they come to choose the parameters they want to use.</p> |
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| | <h2>Improve an existing part</h2> | | <h2>Improve an existing part</h2> |
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| − | <p>We chose to take the existing BBA part of miraculin, and improve it by adding the HXT7 promoter we have already isolated.</p> | + | |
| − | <p>Our new part is HXT7_miraculin - BBa_K2408024 <a href="http://parts.igem.org/ wiki/index.php?title=Part: BBa_K2408024" target="_blank"> http://parts.igem.org/wiki/index.php?title=Part:BBa_K2408024</a> </p> | + | |
| − | <p>HXT7 transcription was repressed at high glucose levels and was detected when the glucose in the culture approached depletion.</p>
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| − | <p>All hexose transporter proteins Hxt2, 4, 6 and 7 in S. cerevisiae are repressed by high glucose concentration, and induced when glucose concentration decreases below a certain level.</p>
| + | <p>We chose to take the existing [<a href="http://parts.igem.org/Part: BBa_K1033121" target="_blank"> BBa_K1033121</a> ] part of miraculin made by team iGEM13_Uppsala and improve it by optimizing it for < i>S. cerevisae</ i> and by adding the ADH1 promoter from [<a href=" http:// parts. igem. org/ Part:BBa_K319005" target="_blank"> BBa_K319005</a> ] of team iGEM10_uOttawa. Our new part is ADH1_Miraculin - [<a href="http:// parts. igem. org/ Part:BBa_K2408023" target="_blank"> BBa_K2408023</ a>]. </ p> |
| − | <p>HXT7 seems to bind glucose with the highest affinity among all glucose transporters, and this fact is associated to a strong induction at low glucose level. The HXT7 promoter region turned out to be suitable for recombinant protein production in yeast and was compared to other yeast promoters (PTEF1, PADH1, PTPI1, PPGK1, PTDH3 and PPYK1) using lacZ as a reporter gene. Among them, PHXT7 was stated as the strongest promoter in continuous culture with limited glucose level.</p>
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| − | <p>
| + | <p>The miraculin glycoprotein, originally produced from the <i>Synsepalum dulcificum</i> plant, is a non-cariogenic substance, with a glycemic index of zero. It is suitable for consumption by diabetics and has the ability to convert sour taste into sweet taste. However, miraculin does not change the taste of the foods / drinks consumed. The mechanism of action of this special protein is still not entirely clear, but it is known that it is triggered in the presence of a low pH. |
| − | <a href=" https:// microbialcellfactories. biomedcentral. com/ articles/10.1186/1475-2859-13-5#CR44" target="_blank"> https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-13-5#CR44</a> | + | Miraculin is a glycoprotein made by plant cells, therefore a yeast like <i>S. cerevisiae</i> would make a better host for expression than bacteria. We optimized the codon of the part for expression in yeast. In addtion, we added few more features: |
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| − | <a href="http:// onlinelibrary. wiley. com/ doi/10.1002/yea.771/pdf" target="_blank"> http:/ /onlinelibrary.wiley. com/ doi/ 10. 1002/ yea. 771/pdf</a > | + | First, we added the ADH1 promoter at the beginning of the sequence. The ADH1 promoter is a strong constitutive promoter, which is used for protein expression in yeast. Originally the ADH1 promoter regulates the expression of the Alcohol Dehydrogenase 1 enzyme in <i>S. cerevisiae</i>. This version of the promoter is a shortened one and it is not inhibited by ethanol. |
| − | </p>
| + | Secondly, we added a histidine tag for purification of the prepared protein and finally added the ADH1 terminator for more effective expression. </p> |
| − | <p>Since transcription of genes with the HXT7 promoter was correlated with the extracellular glucose concentration in the cultures, we can cause the miraculin to be released only when the glucose levels are low and thus adjust the level of its secretion to the desired taste in wine - at a high concentration of glucose it will not be dispensed, and at low concentration it will be secreted and improve the wine's sweet taste.</p>
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During our project we had a great deal of work with different types of promoters - constitutive and inducible. During the planning of the project and the accumulation of knowledge about the various parts, we discovered that much information is lacking about many of these parameters, and the information that exists is scattered and is not arranged in one place. We chose two modulators - TDH3 [BBa_K124002] of team iGEM08_Brown and PGK1 [BBa_K2110012] of team iGEM16_Tianjin, two strong constitutive permutative promoters, and improved their descriptions and the information about them, referring to the main sources we used. We hope that groups coming after us will have faster and more convenient access to information when they come to choose the parameters they want to use.
Improve an existing part
We chose to take the existing [BBa_K1033121] part of miraculin made by team iGEM13_Uppsala and improve it by optimizing it for S. cerevisae and by adding the ADH1 promoter from [BBa_K319005] of team iGEM10_uOttawa. Our new part is ADH1_Miraculin - [BBa_K2408023].
The miraculin glycoprotein, originally produced from the Synsepalum dulcificum plant, is a non-cariogenic substance, with a glycemic index of zero. It is suitable for consumption by diabetics and has the ability to convert sour taste into sweet taste. However, miraculin does not change the taste of the foods / drinks consumed. The mechanism of action of this special protein is still not entirely clear, but it is known that it is triggered in the presence of a low pH.
Miraculin is a glycoprotein made by plant cells, therefore a yeast like S. cerevisiae would make a better host for expression than bacteria. We optimized the codon of the part for expression in yeast. In addtion, we added few more features:
First, we added the ADH1 promoter at the beginning of the sequence. The ADH1 promoter is a strong constitutive promoter, which is used for protein expression in yeast. Originally the ADH1 promoter regulates the expression of the Alcohol Dehydrogenase 1 enzyme in S. cerevisiae. This version of the promoter is a shortened one and it is not inhibited by ethanol.
Secondly, we added a histidine tag for purification of the prepared protein and finally added the ADH1 terminator for more effective expression.
