Part:BBa_K1689006

Coding sequence of FKBP-Cluc394

FKBP-C-luc394 fusion protein ORF

Firefly (Photinus pyralis) luciferase can be split to N-terminal (N-luc) and C-terminal (C-luc) fragments and each of them is inactive. When they two reassembled non-covalently, the enzymatic activity would be reconstituted and the recovered luciferase is able to oxidize luciferin and produce detectable bioluminescence. Currently there are different combinations of split fragments, among which N-luc416/ C-luc398 and N-luc398/ C-luc394 are widely used[1].

FKBP is a monomeric and highly abundant cytosolic protein that serves as the primary receptor for the immunosuppressive ligands FK506 and rapamycin. Previously Raik Gruenberg had already designed the part BBa_J18925, containing the coding sequence of FKBP. Rapamycin-binding domain (FRB) of human mTOR (mammalian Target of Rapamycin) binds with high affinity to FKBP. Rapamycin is able to induce the dimerization to form a FRB-rapamycin-FKBP complex[2]. This protein-protein interaction can be visualized by split luciferase[3]. FRB and FKBP are fused to N-luc and C-luc respectively, and adding rapamycin can induce the approaching and reconstitution of split luciferase (Figure 1a).

2015 Peking iGEM improved the previous part BBa_J18925, they fused C-luc394 to C terminus of FKBP (FKBP-C-luc394, BBa_K1689006) and combined it with N-luc398-FRB (BBa_K1689004) to validate the functional reconstitution of split luciferase. However, compared with N-luc416/ C-luc398, the bioluminescence intensity didn't increase significantly after rapamycin was added (Figure 1). Therefore we discarded them and chose N-luc416/ C-luc398 as our split luciferase in the project (See BBa_K1689003 or BBa_K1689005.)

Figure 1. Rapamycin-induced N-luc-FRB/ FKBP-C-luc complementation. (a) The working mechanism of rapamycin induced dimerization. The interacting protein partners (FRB & FKBP) get closer and dimerize soon after rapamycin is added (40nM) [3], thus to reconstitute the enzymatic activity of luciferase. (b) The experimental data. Error bars denote s.d.; n=3.

References

1. Ramasamy Paulmurugan, Sanjiv S. Gambhir. Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals. Anal Chem. 2005 March 1; 77(5): 1295–1302.

2. Rivera, V. M., T. Clackson, S. Natesan et al. A humanized system for pharmacologic control of gene expression. Nat. Med. 1996. 2:1028–1032.

3. Ramasamy Paulmurugan, Sanjiv S. Gambhir. Combinatorial Library Screening for Developing an Improved Split-Firefly Luciferase Fragment-Assisted Complementation System for Studying Protein-Protein Interactions. Anal. Chem. 2007, 79, 2346-2353.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 662
  Illegal AgeI site found at 801
  
• 1000
  COMPATIBLE WITH RFC[1000]