Team:Dundee/Bacterial Two Hybrid
To characterise our protein-protein interactions, we will use the bacterial two hybrid system. The bacterial two hybrid system exploits the modular properties of the adenylate cyclase to monitor protein-protein interactions. The catalytic domain of adenylase cyclase consists of two complementary fragments, T18 and T25 and catalyse the formation of cyclic AMP from ATP. These fragments can be physically separated but they will no longer be able to catalyse the synthesis of cyclic AMP. To test whether two proteins X and Y interact, on can be fused to the N terminal T25 fragment and the other to the C terminal T18 fragment. If the two proteins interact, T25 and T18 will be brought into close proximity and reconstitute an active adenylate cyclase which can then produce cyclic AMP. Cyclic AMP can then bind to the catabolite activator protein to form a complex. This complex is a transcription factor that activates the expression of several catabolic genes. One example of a reporter gene that we might use is the lacz gene encoding the enzyme beta galactosidase. Active β-galactosidase may be detected by X-gal, which produces a characteristic blue dye when cleaved by β-galactosidase. So bacteria expressing interacting proteins will form blue colonies on rich LB medium supplemented with x-gal. Whereas bacteria expressing non interacting proteins will remain white.
Figure 1 - The catalytic domain of adenylase cyclase consists of two complementary fragments, T18 and T25 and catalyse the formation of cyclic AMP from ATP. These fragments can be physically separated but they will no longer be able to catalyse the synthesis of cyclic AMP.
Figure 2 - To test whether two proteins X and Y interact, on can be fused to the N terminal T25 fragment and the other to the C terminal T18 fragment. If the two proteins interact, T25 and T18 will be brought into close proximity and reconstitute an active adenylate cyclase which can then produce cyclic AMP.
Figure 3 - Cyclic AMP can then bind to the catabolite activator protein to form a complex. This complex is a transcription factor that activates the expression of several catabolic genes.
