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Two-Hybrid System

In order to make a great screening for peptide aptamers you need a well-functioning screening system. Our screening system is the bacterial two-hybrid system. This is system is based on the reconstitution of the adenylate cyclase and allows detection of protein-protein interaction. In this page we validate the function of the two-hybrid system.

Control Experiment

To validate that our T18 and T25 domain constructs in fact can be used to study protein-protein interactions, we made a control experiment, where the leucine zipper region from the GCN4 yeast protein was fused to the T18 and T25 domains (T18-Zip+T25-Zip). Leucine zippers are known to interact by forming homodimers. If the system indeed works, their interaction will lead to functional complementation between the T18 and T25 domains. This leads to the synthesis of cAMP. By using a cAMP-induced reporter system, one can observe whether or not there is an interaction.
When working with the bacterial two-hybrid system, it is necessary to use a strain with a deficiency in the gene encoding the adenylate cyclase, cyaA. For this purpose we constructed the Escherichia coli K12-strain, MG1655 ΔcyaA. We also used the cyaA-deficient E. coli K12-strain BTH101 (MC1061-derived). This was for the purpose to exploit the lacZ-derived reporter system. The lacZ gene encodes a β-Galactosidase which is positively controlled by cAMP. Our goal was to use the RFP reporter system in the MG1655 ΔcyaA-strain. However, this control experiment was carried out in the BTH101-strain.
Four different combinations were sequentially co-transformed into the BTH101-strain: pSB1C3-T18+pSB1K3-T25 pSB1C3-T18+pSB1K3-T25-Zip pSB1C3-T18-Zip+pSB1K3-T25 pSB1C3-T18-Zip+pSB1K3-T25-Zip