Part:BBa_K1997017
P+R->sGFP-N->FRB->RBS->FKBP ->sGFP-C->TER
Usage and Biology
Since protein-protein interactions (PPIs) have been reported to play important roles in signal transduction and gene expression, methods for monitoring PPIs in cells have been developed rapidly for years1 . Among which, split-GFP system, due to its wide applicability, was widely applied in various fields of researches 2 .
Special Design
As a member of the collection PPI tool kit, special designs were taken for to optimize the applicability and adaptive of such parts. Specifically, a novel designed substitution system, through which, two proteins could be fused with their corresponding split-GFP fragment at the same time using Golden-Gate Assembly, was introduced to dramatically simplify the cloning process).
Figure 1. Schematic representation of the workflow of the substitution system
Coding sequence of proteins to be studied can be assembled with a RBS in between, a PCR procedure adding a 5’-ATAGGGGAGACC-3’ flank to the sense strand and a 3’-TCCAGAGTCAAA-5’ flank to the anti-sense would make it a proper substrate for the BsaI nuclease digest. Once digested, the product could be ligated together with the BsaI treated BBa_K1997017 to form a brand new device expressing the proteins of sGFP-N-Protein1, Protein2-sGFP-C and. The interaction between Protein1 and protein 2 could then be determined through the green florescent intensity.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1381
Illegal BsaI.rc site found at 736
Illegal BsaI.rc site found at 1595
Experimental Validation
This part is validated through four ways: enzyme cutting, PCR, Sequence, and functional testing
Sequencing
This part is sequenced as correct after construction.
PCR
Methods
The PCR is performed with Premix EX Taq by Takara.
F-Prime: 5’- GAATTCGCGGCCGCTTCTAGAATGC-3’
R-Prime: 5’- GGACTAGTATTATTGTTTGTCTGCC-3’
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu. The result of the agarose electrophoresis was shown on the picture below.
Enzyme digestion test
Methods
After the assembly ,the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with EcoRI and SpeI restriction endonuclease bought from TAKARA.
The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours.
The Electrophoresis was performed on a 1％ Agarose glu.
The result of the agarose electrophoresis was shown on the picture above.
Functional Test
Building on the results of BBa_K1997015 and BBa_K1997016, further experiments were conducted to demonstrate the imbedded substitution system. For such matters, BBa_K1997017 was constructed by replacing the Zif268 region in BBa_K1997015 into a “FRB-RBS-FKBP” fragment. The cloning results were validated through sequencing.
For function validation, we used Rapamycin to induce the interaction between FRB and FKBP. For such assay, E.coli carrying respective plasmid was cultured overnight under IPTG induction. Cells were then collected and lysed by high-pressure homogenizer. Once lysed, 0.4nM of Rapamycin was added into the cell lysate to induce the protein-protein reaction Fluorescence intensity measured right after the addition of Rapamycin showed a significant improvement on relative FI, thus validated the function of this part.
Figure 1. Rapamycin-induced sGFP-N-FRB/sGFP-C-FKBP interaction. (A) Schematic representation of the rapamycin induced protein-protein interaction. The adding of rapamycin would induce the interaction between FRB and FKBP, thus shortened the range between split-GFP fragments and reconstruct its structure for fluorescence generation. (B) Fluorescent assay showing the fluorescent intensity with/without Rapamycin induction. Relative FI was calculated with normalization of the OD600 value. For Fold change Relative FI, relative FI of the group without Rapamycin induction was set arbitrarily as 1.0, and the levels of the other groups were adjusted correspondingly. The concentration of Rapamycin used in the experiment was 40nM. This experiment was run in three parallel reactions, and the data represent results obtained from at least three independent experiments. **p<0.01.
References
[1] Day, R. N. & Davidson, M. W.The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38, 2887-2921, doi:10.1039/b901966a (2009).
[2] Pfleger, K. D.& Eidne, K. A. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nature methods 3,165-174, doi:10.1038/nmeth841 (2006).
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