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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

These transformations were plated out on LB/X-gal plates with appropriate antibiotics and 40 µg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside).

Streaks of BTH101 on LB/X-gal plates, containing pSB1C3-T18 + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25 and pSB1C3-T18-Zip + pSB1K3-T25-Zip.

As expected, the results only showed complementation between T18 and T25 when the leucine zipper was fused to both of the domains. These results prove that our constructs function as expected, and that it can be used to detect protein-protein interactions. This indicates that the system can be used to screen for peptide aptamers.

If we wanted to use the RFP reporter system instead, the same experiment could be conducted and red colonies should be observed instead of blue. The transformations should however be plated out on LB plates without X-gal.

Expression

GFP was fused to the two components of the two-hybrid system, T18 and T25. This allows us to detect expression of these two components that are both under control of a lac promoter. Additionally, the presence of green fluorescence will verify that proteins can be fused to T18 and T25, and still fold into the correct structure.

The two constructs pSB1C3-T18-GFP and pSB1C3-T25-GFP were transformed into the E. coli K12-strain TOP10. The following fluorescence microscopy images confirm the presence of green fluorescence. This means that both T18 and T25 are expressed under the lac promoter, and that proteins fused to these two constructs will fold into the correct structure.