Team:SDU-Denmark/Tour31
Two-Hybrid Screening
We are using a Two-Hybrid system to screen for aptameres as an alternate to antibodies. It can be used for detecting protein-protein interactions, by measuring levels of cAMP. The way a two-hybrid system functions is:
Figure 1: T18 and T25 each contain one of the coding sequences for the two catalytic subunits of adenylate cyclase.
The subunits of the adenylate cyclase T18 and T25 functions only when close together. The linker ensures this. The cyclase is then able to convert ATP to cAMP.
Our system
In our Two-Hybrid screening system, the linker between T18 and T25 is; A scaffold protein, coupled to T18, with the aptamer in its active site and on T25 a small linker domain followed by the target protein. To detect an increase in cAMP levels, a MG1655:delta-CyA is used. Detection is insured by a PstA a cAMP-activated promotor. The product of transcription is RFP (red fluorescent protein) visible by the eye and with fluoresces. The stronger the interaction between aptamer and target, the stronger the fluorescent. Selection of high affinity aptameres is thereby enabled.
Figure 2:
The subunits of the adenylate cyclase T18 and T25 functions only when close together. The linker ensures this. The cyclase is then able to convert ATP to cAMP. After selection, the specific sequence for an aptamer + linker and scaffold is cut out and transferred, to a another plasmid containing intein, used for production. This colony has the …. system promoting secretion of our product. As part of the construct is the intein, which is self cleaving used for purification of product in an EBA.