Part:BBa_K1689006

Coding sequence of FKBP-Cluc394

FKBP-Cluc394 fusion protein ORF

Firefly (Photinus pyralis) luciferase can be split to N-terminal (Nluc) and C-terminal (Cluc) 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 Nluc416/ Cluc398 and Nluc398/ Cluc394 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 Nluc and Cluc 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 Cluc394 to C terminus of FKBP (FKBP-Cluc394, BBa_K1689006) and combined it with Nluc398-FRB (BBa_K1689004) to validate the functional reconstitution of split luciferase. However, compared with Nluc416/ Cluc398, the bioluminescence intensity didn't increase significantly after rapamycin was added (Figure 1). Therefore we discarded them and chose Nluc416/ Cluc398 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]