Difference between revisions of "Part:BBa K1638018:Design"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1638018 short</partinfo>
 
<partinfo>BBa_K1638018 short</partinfo>
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<b>Library</b>
 
<b>Library</b>
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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 is A,T,G or C and K is G or C). When designing you library, it should be flanked by two XhoI res. sites. This enables insertion into the xhoI res. site intended for the library insertion.  
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<ul>
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    <li>Choose the size of your library. Typically the length of the variable peptide loop is between 10 and 20 amino acids. </li>
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    <li>Dependent on the size your variable peptide loop, you can for example contruct the library as following: ATATGCGCTCGAG(NNK)<sub>x</sub>GAGCTCATTACCG (where N represent A, T, G or C and K represent G or C). The NNK codon minimizes the number of stop codons. The XhoI restriction included allows insertion of library into the scaffold.</li>
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    <li>Order the library as duplex-DNA</li>
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    <li>When delivered, cut the library and scaffold with XhoI restriction enzyme and ligate.</li>
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</ul>
  
 
<b>Important note</b>: This part cannot be used in the standard backbones pSB1C3 or pSB1K3, as they include a xhoI restriction site. Instead we recommend using the [https://parts.igem.org/Part:pSB1A2 pSB1A2] backbone
 
<b>Important note</b>: This part cannot be used in the standard backbones pSB1C3 or pSB1K3, as they include a xhoI restriction site. Instead we recommend using the [https://parts.igem.org/Part:pSB1A2 pSB1A2] backbone
  
 
===Source===
 
===Source===
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pUT18C
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.
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<br>
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Human thioredoxin sequence: UniProt (AC: P10599)
  
 
===References===
 
===References===
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[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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<br>
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[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.

Latest revision as of 10:05, 18 September 2015

hTrx-based scaffold fused to T18 through a flexible linker


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]


Design Notes

This part was synthesized by IDT as a gBlock gene fragment.


Library

  • Choose the size of your library. Typically the length of the variable peptide loop is between 10 and 20 amino acids.
  • Dependent on the size your variable peptide loop, you can for example contruct the library as following: ATATGCGCTCGAG(NNK)xGAGCTCATTACCG (where N represent A, T, G or C and K represent G or C). The NNK codon minimizes the number of stop codons. The XhoI restriction included allows insertion of library into the scaffold.
  • Order the library as duplex-DNA
  • When delivered, cut the library and scaffold with XhoI restriction enzyme and ligate.

Important note: This part cannot be used in the standard backbones pSB1C3 or pSB1K3, as they include a xhoI restriction site. Instead we recommend using the pSB1A2 backbone

Source

pUT18C
Human thioredoxin sequence: UniProt (AC: P10599)

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.