Part:BBa_K1638011
Leucine zipper fused to T18 domain of cyaA from Bordetella pertussis
This part consist of the leucine zipper region of the yeast GCN4 protein fused to the T18 domain as used in the bacterial two-hybrid system [1]. This part allows test of functional complementation between the T18 and T25 domain. When T18-zip and T25-zip are co-transformed, the homodimerisation of the two leucine zipper will cause association of the T18 and T25 domain and catalyse formation of cAMP. By using a cAMP-induced reporter system, one can detect correct complementation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 711
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 252
Illegal NgoMIV site found at 662
Illegal AgeI site found at 468 - 1000COMPATIBLE WITH RFC[1000]
Characterization
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 the cAMP-induced lacZ reporter system, one can observe whether or not there is an interaction. This system is part of the cyaA-deficient Escherichia coli K12-strain BTH101 (MC1061-derived). The lacZ gene encodes a β-Galactosidase which is positively controlled by cAMP.
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 (chloramphenicol 25 µg/ml and kanamycin 25 µg/ml) and 40 µg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). X-gal produces a blue dye, when cleaved by β-Galactosidase. This will give a characteristic blue phenotype.
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 leucine zippers form homodimers, and that our T18/T25 constructs function as expected. This indicates that the system indeed can be used to study protein-protein interactions.
¤Note: all of the constructs were under control by lac promoter, Plac.
References
[1]: Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(10):5752-6.
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