Team:GreatBay United/parts

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Parts

GreatBay_United used the part anti-GFP, their inner structure of genes do not contain the recognition site of type IIS endonuclease and can be used for Goldengate gene assembly. As a reporter gene, GFP is commonly used in synthetic biology. AntiGFP can be fused with GFP, and the addition of restriction sites in GFP and anti-GFP can be used to test the enzyme activity. And GreatBay_United successfully tested the enzyme activity of the protease HCV with anti-GFP.

(anti-GFP expression cassette)
In previous IGEM teams, some of which used this part. This year, GreatBay_United tried to display this antibody onto the yeast surface. In our project, we used this part and characterized it as well as tested it.
There are two kinds of construction: Solely expressing the antibody or fusion expression with GFP. To display onto surface, an existing part (Aga2) was used to lead the recombination protein to the cell membrane.

(the cassette of yeast surface display of GFP-HCV protease cut site-antiGFP complex)
For the fusion expression version, the GFP part and anti-GFP part were linked with HCV protease cutting site. Thus, if both complex and antiGFP were successfully displayed on the surface, they will be able to sense the HCV NS3 protease in solution. The protease would cut and release the antiGFP linked to GFP, and the solely expressed anti-GFP would had chance to connect with GFP. this interaction would lead to the macroscopical agglutination phenomenon when we mix two kinds of engineered yeast together and also HCV protease.

(the two types of yeast co-culture with HCV protease in the medium)

(the two types of yeast co-culture without HCV protease in the medium)
Because there were different affinity of antiGFPs, it could be several combination choices to design. To figure out which one can work best, we constructed two kinds GFP-antiGFP(H/L) and two kinds antiGFP(H/L), then mixed them for their full combinations, thus there are 4 types.

(all 4 combinations)

(4 combinations results without HCVp added)

(4 combinations results with HCVp added)