Part:BBa_K1638018
hTrx-based scaffold fused to T18 through a flexible linker
This composite part consists of the hTrx-scaffold [1] (used for presenting peptide aptamers) fused to the T18 domain of CyaA (used in the bacterial two-hybrid system). This part is used in bacterial two-hybrid screening [2] for peptide aptamers against specific targets. When a target candidate is fused to the T25 domain of CyaA, and a peptide aptamer binds the target, the T18 and T25 domain is brought into proximity of one another and the formation of cAMP is induced. When combined with a cAMP reporter system, like a system promoting the transcription of RFP, the presence of red-flourescent bacteria indicates an interaction between the peptide aptamer and the target protein.
The Library
In oder to use this part for peptide aptamer screening, a random nucleotide library has to be inserted into the scaffold. This library codes for a random peptide library that enables screening of a diverse set of peptide aptamers with different specificity. Typically, the library consist of 20 sets of the codon NNK (where N is A,T,G or C and K is G or C). See design for further notes on design of library.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 624
Illegal XhoI site found at 753 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 165
Illegal NgoMIV site found at 575
Illegal AgeI site found at 381 - 1000COMPATIBLE WITH RFC[1000]
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. [2]: Borghouts C, Kunz C, Delis N, Groner B. Monomeric Recombinant Peptide Aptamers Are Required for Efficient Intracellular Uptake and Target Inhibition. Molecular Cancer Research. 2008;6(2):267-81.
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