Part:BBa_K1638012
Leucine Zipper
This part contains the leucine zipper region of the GCN4 protein from Saccharomyces cerevisiae. Leucine zippers is a common DNA-binding motif that is known to form a homodimer of two alfa-helix monomers.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 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.
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