Difference between revisions of "Part:BBa K1638018"
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This composite part consists of the [https://parts.igem.org/Part:BBa_K1638014 hTrx-scaffold] (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 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.
 
This composite part consists of the [https://parts.igem.org/Part:BBa_K1638014 hTrx-scaffold] (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 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.
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The Library
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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 equals A,T,G or C and K equals G or C). See design for further notes on design of library.
  
 
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Revision as of 17:29, 10 September 2015

hTrx-based scaffold fused to T18 through a flexible linker

This composite part consists of the hTrx-scaffold (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 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 equals A,T,G or C and K equals G or C). See design for further notes on design of library.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 624
    Illegal XhoI site found at 753
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 165
    Illegal NgoMIV site found at 575
    Illegal AgeI site found at 381
  • 1000
    COMPATIBLE WITH RFC[1000]