Difference between revisions of "Part:BBa K1638005:Design"

(→‎Source)
(→‎References)
 
(One intermediate revision by the same user not shown)
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
Addition of a protein coding domain to the suffix by Standard Assembly (RFC[10]) creates a scar-site. As the scarsite encodes a stop codon (uacuag), this will leave the fused protein coding domain untranslated.  
+
Addition of a protein coding domain to the suffix by Standard Assembly RFC[10] creates a scar-site. As the scarsite encodes a stop codon (uacuag), this will leave the fused protein coding domain untranslated.  
  
 
Instead we recommend the following assembly method:
 
Instead we recommend the following assembly method:
Line 27: Line 27:
  
 
===References===
 
===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.

Latest revision as of 00:28, 18 September 2015


T18 domain of cyaA from Bordetella pertussis (IPTG inducible)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 711
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 252
    Illegal NgoMIV site found at 662
    Illegal AgeI site found at 468
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Addition of a protein coding domain to the suffix by Standard Assembly RFC[10] creates a scar-site. As the scarsite encodes a stop codon (uacuag), this will leave the fused protein coding domain untranslated.

Instead we recommend the following assembly method:

1) Design primers for amplification of the C-terminal fusion protein with BamHI resitriction site included in the forward primer. The primers must also include prefix and suffix.

1.2) Forward primer: 5'-ATATGGATCCNNN...NNN-3'

1.3) Reverse primer: 5'-ATATCTGCAGCGGCCGCTACTAGTANNN..NNN-3'

2) Amplify through PCR with designed primers

3) Digest PCR-product and pSB1C3-T18 with BamHI and PstI.

4) Ligate the two digested product.

Source

pUT18C

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.