BRET Vectors
Three sets of BRET vectors have been made (see Table):
- pBS-35S-RLUC and pBS-35S-YFP allow you to make plain fusions to either the N-terminus or C-terminus of RLUC (Renilla luciferase) or YFP (yellow fluorescent protein). Cite Subramanian et al., 2004. PNAS 101:6798-6802.
- A second set of vectors features short stretches of alanines as spacers between the fusion partner and the BRET tag.
- Gateway vectors with and without T-DNA borders. Cite Subramanian et al., 2006. Plant Journal 48:138-152.
We recommend using the human-codon-optimized version of RLUC (hRLUC).
As is typical with fusions to fluorescent proteins, some of our RLUC fusions give high luciferase activity and others don't. Small proteins (<30kDa) are more likely to retain high activity than large ones (>60kDa). Fusions to the N-terminus and the C-terminus can work equally well. A ‘good’ RLUC fusion will give activity 100-fold above the background noise of the instrument (and ‘bad’ fusions are almost inactive).
Note: We also have additional fluorescent protein expression vectors for tagging with GFP (Cite von Arnim et al., 1998. Gene 221:34-43) and CFP.
Vectors for expressing protein fusions to Renilla luciferase (RLUC) or yellow fluorescent protein (YFP). All the vectors listed here are based on Bluescript (ampicillin resistance), pPZP222 (spectinomycin resistance) or pBIN19 (resistance to kanamycin and chloramphenicol). If not mentioned otherwise, LUC or YFP are driven by the 35S promoter with a double enhancer, and a 35S terminator.
Table: Expression vectors for BRET tagging in plants
| Plasmid | Tag * |
Bacterial Resistance |
Genbank Accession |
Stock No. | Remarks |
| RLUC | N/C | Amp | AY189980 | 476 | Renilla Luciferase |
| hRLUC | N/C | Amp | Sequence | 696 | (h)humanized Codon usage |
| YFP | N/C | Amp | AY189981 | 554 | EYFP |
| Alanine linker vectors | |||||
| Ala-RLUC RLUC-Ala |
C N |
Amp
Amp |
AY189982 AY189983 |
739 740 |
Linker: AAAPVAAAAAA AAAARS |
| Ala-YFP YFP-Ala |
C N |
Amp Amp |
AY189984 AY189985 |
741 742 |
|
| Vectors for facile directional insertion of native promoters | |||||
| RLUC mut5’ YFP mut5’ |
N/C N/C |
Amp Amp |
Sequence Sequence |
776 747 |
|
| Gateway destination vectors for transient expression | |||||
| RLUC attR hRLUC-attR |
N N |
Amp Amp |
AY995136 AY995138 |
797 923 |
|
| YFP attR attR-RLUC |
N C |
Amp Amp |
AY995137 AY995139 |
798 1270 |
|
| attR-hRLUC attR-YFP |
C C |
Amp Amp |
AY995140 AY995141 |
1273 804 |
|
| Gateway T-DNA vectors | |||||
| hRLUC-attR attR-hRLUC |
N C |
Spec Spec |
AY995142 AY995143 |
947 1429 |
Mas:bar** Mas:bar** |
| YFP-attR attR-YFP |
N C |
Kan/chl Kan/chl |
AY995144 AY995145 |
961 963 |
Neo:nptll Neo:nptll |
| Control plasmid | |||||
| RLUC-YFP hRLUC-YFP |
Amp Amp |
Sequence Sequence |
560 1328 |
Positive control Positive control |
|
| Gaussia luciferase (18kDa) vectors | |||||
| GLUC hGLUC |
N/C N/C |
Amp Amp |
Sequence | 635 636 |
|
| hGLUC mut5’ | N/C | Amp | 789 | ||
| ‘Weak promoter’ vectors | |||||
| RLUC-YFP RLUC RLUC |
N/C N/C N/C |
Amp Amp Amp |
822 823 868 |
35S-90 35S-90 No TEV leader |
|
| GFP vectors | |||||
| GFP CFP GFP5 |
N/C N/C N/C |
Amp Amp Amp |
AF078810 |
319 574 393 |
Siemering et al. (1996) Curr. Biol. 6, 1653-1663 |
-> * Tag will appear on the N-terminus or C-terminus of your protein.
-> ** Restriction with XbaI and BglII indicates additional cleavage sites, which lie outside of the 35S:hRLUC-attR cassette. These do not appear to interfere with recombination or gene expression.
-> Ampicillin/Carbenicillin, 100 mg/L; Kanamycin, 50 mg/L; Spectinomycin, 100 mg/L
-> Chloramphenicol (20mg/L), is used together with kanamycin, the latter as an auxiliary selection.
-> mas:bar and neo:nptII are Basta and kanamycin resistance selection markers on the T-DNA.
updated: October 5, 2007