Our team aimed at adjusting the expression level of any existing promoter. Thus, we modified the Kozak sequence of the promoter in the plasmid BBa_K775004, a part from the University of the TU-Delft 2012 iGEM team. Our goal is to reduce the expression efficacy of this vector, and make it suitable to express a suicide gene for information destruction. To reduce the expression efficiency of this part in yeast, we modified the Kozak sequence by mutating the sequence “AAAAAA” to “GCCACC”. After that, our information destruction system was built and kill the information host yeast in a desired period of time. See more details in the basic part BBa_K2542014. We used the enhanced green fluorescent protein (EGFP) to test the effect of this modification. The generated plasmid allows a type yeast to sense the factor secreted by α-type yeast and express EGFP. Thus, when the promoter was activated by factor secreted by α-type yeast, the EGFP would be expressed. Thus, the EGFP expression would be the standard to tell the strength of our improved promoter.
Our team attempted to verify the strengthen of a promoter called Fig2c in a part of BBa_K1829002 from the iGEM 2015 UCSF team. For this purpose, we inserted the pFig2c promoter and EGFP coding sequence into the pesc-ura plasmid, and transferred it into a-type saccharomyces cerevisiae.The expression of the Fig2c promoter was induced by adding α mating factor. When the Fig2c promoter is induced, EGFP is expressed. In this way, we can detect the strengthen of the Fig2c promoter using EGFP as a reporter.
This part serves as a lock structure in our scheme, in which EGFP is used as a substitute for our encrypted information, and can be used to verify the function of our lock part. Thus, only when a specific small RNA binds to the stem loop and changes the stem loop secondary structure, EGFP transcription can be initiated and EGFP expression can be achieved. This unit contains six parts: the CYC promoter, STEMLOOP, EGFP coding sequence, the CYC terminator and homology arms HA1 and HA2. HA1 and HA2 act as homology arms to achieve homologous recombination, which can insert the DNA with the lock structure sequence into the yeast genome. The stem loop acts as a lock to protect our encrypted information to be transmitted.
This part serves as a key structure in our scheme. This plasmid expresses a small RNA that can specifically bind to the secondary structure of the corresponding stem loop and resolve its structure, which allows the DNA sequence downstream the stem loop to be transcribed.
This plasmid allows a-type yeast to sense the pheromone α factor secreted by α-type yeast. The a-type Saccharomyces cerevisiae expresses Bax protein, which can arrest cell cycle and reduce cell density. Therefore, upon induction by α factor the a-type yeast cells carrying this plasmid undergo a decrease in cell density, which can be used to protect our encrypted information.