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| − | We chose to take the existing [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1033121 BBa_K1033121] 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K319005 BBa_K319005] of team iGEM10_uOttawa. Our new part is ADH1_Miraculin - [http://parts.igem.org/wiki/index.php?title=Part:BBa_K2408023 BBa_K2408023]. | + | 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>]. |
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| − | This is a part improvment from BBa_K1033121 - team Uppsala 2013
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| − | Miraculin optimized for S. cerevisae with the ADH1 promoter and histidine tag.
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| − | 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 low pH. | + | 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. |
| − | Because miraculin is a glycoprotein made by plant cells, therefore a yeast like S. cerevisiae would make a better expression host than bacteria. We codon optimize the part for yeast expression. In Addtion, we added few more feature: | + | Because miraculin is a glycoprotein made by plant cells, therefore a yeast like <i>S. cerevisiae</i> would make a better expression host than bacteria. We codon optimize the part for yeast expression. In Addtion, we added few more feature: |
| − | First, we added the ADH1 promoter at the beginning of the sequence. The ADH1 promoter is a strong constitutive promoter, that uses for protein expression in yeast. Originally the ADH1 promoter regullate the expression of the Alcohol Dehydrogenase 1 enzyme at Saccharomyces Cerevisiae. This version of the promoter is a shortened one, registered by team XXXXX, and it is not inhibited by ethanol. | + | <br> |
| | + | First, we added the ADH1 promoter at the beginning of the sequence. The ADH1 promoter is a strong constitutive promoter, that uses for protein expression in yeast. Originally the ADH1 promoter regullate the expression of the Alcohol Dehydrogenase 1 enzyme at <i>Saccharomyces Cerevisiae</i>. This version of the promoter is a shortened one, and it is not inhibited by ethanol. |
| | Second, we added a histidine tag for purification of the prepared protein, and finally added the ADH1 terminator for more effective expression at the end. | | Second, we added a histidine tag for purification of the prepared protein, and finally added the ADH1 terminator for more effective expression at the end. |
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| | <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> | | <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 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 [BBa_K124002] of team iGEM08_Brown and PGK1 [BBa_K2110012] 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.
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.
Because miraculin is a glycoprotein made by plant cells, therefore a yeast like
S. cerevisiae would make a better expression host than bacteria. We codon optimize the part for yeast expression. In Addtion, we added few more feature:
First, we added the ADH1 promoter at the beginning of the sequence. The ADH1 promoter is a strong constitutive promoter, that uses for protein expression in yeast. Originally the ADH1 promoter regullate the expression of the Alcohol Dehydrogenase 1 enzyme at
Saccharomyces Cerevisiae. This version of the promoter is a shortened one, and it is not inhibited by ethanol.
Second, we added a histidine tag for purification of the prepared protein, and finally added the ADH1 terminator for more effective expression at the end.
