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.