Difference between revisions of "Part:BBa K1689003"

Latest revision as of 15:28, 27 September 2015

Coding sequence of Nluc416-FRB

N-luc416-FRB 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].

Rapamycin-binding domain (FRB) of human mTOR (mammalian Target of Rapamycin) binds with high affinity to FK-506-binding protein 12 (FKBP). Previously Raik Gruenberg had already designed the part BBa_J18926, containing the coding sequence of FRB. 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_J18926, they fused N-luc416 to N terminus of FRB (N-luc416-FRB, BBa_K1689003) and combined it with FKBP-C-luc398 (BBa_K1689005) to validate the functional reconstitution of split luciferase. The result below (Figure 1b) confirmed that the luciferase activity is able to be successfully reconstituted in a rapamycin-dependent manner.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1689003 short</partinfo>
-Nluc 416-FRB fusion protein ORF
+N-luc416-FRB 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 Nluc 416 / Cluc 398 and Nluc 398/ Cluc 394 are widely used[1].
+Firefly (<I>Photinus pyralis</I>) 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].
-Rapamycin-binding domain (FRB) of human mTOR (mammalian Target of  Rapamycin) binds with high affinity to FK-506-binding protein 12 (FKBP). Previously Raik Gruenberg had already designed the part [https://parts.igem.org/Part:BBa_J18926 BBa_J18926], containing the coding sequence of FRB. 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).
+Rapamycin-binding domain (FRB) of human mTOR (mammalian Target of  Rapamycin) binds with high affinity to FK-506-binding protein 12 (FKBP). Previously Raik Gruenberg had already designed the part [https://parts.igem.org/Part:BBa_J18926 BBa_J18926], containing the coding sequence of FRB. 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).
-Peking iGEM improved the previous part [https://parts.igem.org/Part:BBa_J18926 BBa_J18926], they fused Nluc 416 to N terminus of FRB (Nluc 416-FRB, BBa_K1689003) and combined it with FKBP-Cluc 398 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1689005 (BBa_K1689005)] to validate the functional reconstitution of split luciferase. The result below (Figure 1b) confirmed that the luciferase activity is able to be successfully reconstituted in a rapamycin-dependent manner.
+Peking iGEM improved the previous part [https://parts.igem.org/Part:BBa_J18926 BBa_J18926], they fused N-luc416 to N terminus of FRB (N-luc416-FRB, BBa_K1689003) and combined it with FKBP-C-luc398 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1689005 (BBa_K1689005)] to validate the functional reconstitution of split luciferase. The result below (Figure 1b) confirmed that the luciferase activity is able to be successfully reconstituted in a rapamycin-dependent manner.