Team:NEFU China/Improve

Improvement Parts

Improvement parts

Number Name Description
BBa_K2542014 pFUS1-EGFP-tCYC

Our team were 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.

We creat a new parts BBa_K2542014,which is based on BBa_K775004. BBa_K2542014 is more suitable for somewhere which needs low expression in yeast.It's allows a type yeast to sense the α factor secreted by α-type yeast and express green fluorescent protein. We modified the part BBa_K775004 of the previous team by turning mutating the sequence “AAAAAA” into “GCCACC” to make the expression efficiency weaker,and make it more appropriate for suicide gene(Information Destroyed ). We constructed two plasmids with different kozak sequences and transferred them into yeast. Then, we use SD-Trp medium to culture yeast, and add with the solution which containing α factor. The solution containing α factor is obtain by extracting the liquid supernatant of the culture medium for culturing α-type yeast. After we add 200ul the α factor sloution to each one and culture yeast for 20 hours,we measured the expression of GFP in yeast by flow cytometry.