Part:BBa_I715021:Design

pLac:RBS.1:GFP1:hix:GFP2

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 916
  

Design Notes

We designed 4 primers to split GFP in half. They are shown in the table below, with colors to help you see the subsections within the primers.

Once the PCR products were cloned and sequenced, we knew they would be ligated together with a hix site. This figure shows you the two scars, the hix DNA, and the single base to maintain the reading frame.

Here are the encoded amino acids when the hix is inserted between GFP1 and GFP2. * indicate hydrophobic amino acids.

Once the subparts are ligated together, you woud see this hixC sequence inserted between the two halves of GFP (scars not shown).

Finally, this cartoon helps you see the final construct that actually works. Part BBa I7159 is in pSB1A2 plasmid.

Source

This work was conducted by Karen Acker, 2007 graduate of Davidson College. She summarized her work [http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/karen_project.html at this web site.] Included in this guide, is a Jmol tutorial [http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/GFP_halfframe.html at this URL.]

We have now automated this process of gene splitting. You can use [http://gcat.davidson.edu/iGEM07/genesplitter.html our web page] to make it much easier. It will tell you which primers to use if you know where to make the cut.

References

1. Abedi MR, Caponigro G, and Kamb A. Green fluorescent protein as a scaffold for intracellular presentation of peptides. Nucl. Acids Res. 26: 623-630 (1998).
2. Cabantous S, Terwilliger TC, and Waldo GS. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature biotechnology. 23: 102-107 (2004).
3. Hu CD and Kerppola TK. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nature Biotechnology. 21: 539-545 (2003).
4. Magliery TJ and Regan L. Reassembled GFP: detecting protein-protein interactions and protein expression patterns. Green Fluorescent Protein: Properties, Applications, and Protocols, 2nd ed. Eds. Martin Chalfie and Steven R. Kain. 2006.
5. Magliery TJ, Wilson CGM, Pan W, Mishler D, Ghosh I, Hamilton AD, and Regan L. Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. J. Am. Chem. Soc. 127:146-157 (2005).
6. Paramban RI, Bugos RC, and Su WW. Engineering green fluorescent protein as a dual functional tag. Biotechnology and Bioengineering. 86.6: 687-697 (2004).
7. Shyu YJ, Liu H, Deng X, Hu CD. Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. BioTechniques. 40: 61-66 (2006).