Part:BBa_K916002:Design

TraR-CGFP Translational Fusion

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 984
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 910
  

Design Notes

TraR was cloned into existing sites in the pMRBAD-link-NGFP plasmid (NcoI and SphI). Because it was a translational fusion, care had to be taken to ensure the protein was in frame. Also, the pMRBAD-link-CGFP plasmid was designed to use NcoI and AatII for cloning, but TraR contained two AatII sites. Cloning into the SphI site instead added two amino acids to the linker, one of which was a cysteine. It is unclear whether these additional amino acids will disrupt the ability for TraR and the CGFP fragment to fold independently from one another.

The junction between traR and the C-terminal GFP fragment cloned into the SphI site:

                   SphI          AatII
   R   K   L   I   A   C   N   G   T   S   G
   cgg aaa ctc atc gca tgc aat ggg acg tcg ggt 
       <-- traR--| |-- linker -->      

Source

TraR came from a plasmid (pCF222) provided by [http://www.bio.indiana.edu/faculty/directory/profile.php?person=cfuqua Dr. Clay Fuqua] at University of Illinois Champaign-Urbana. The CGFP fragment came from a plasmid (pMRBAD-link-CGFP) provided by [http://www.yale.edu/reganlab/ Dr. Lynne Regan] at Yale University.

References

Wilson CGM, Magliery TJ, Regan L. 2004. Detecting protein-protein interactions with GFP-fragment reassembly. Nat Methods 1: 255. [http://www.yale.edu/reganlab/pdfs/WilsonRegan2004NatMethods.pdf PDF.]