Part:BBa_K1997016
P+R->sGFP-N->Zif-N->RBS->Zif-C->sGFP-C->TER
This part is an integrated tool for protein-protein interaction research using split-GFP system as reporter. the "Zif-N->RBS->Zif-C" subpart can be easily replaced using Golden Gate technique with BsaI
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_K1997016 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]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 832
Illegal AgeI site found at 916
Illegal AgeI site found at 1036 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1039
Illegal BsaI.rc site found at 736
Illegal BsaI.rc site found at 1253
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
This part was tested together with K1997015 using K1997016 as control.
To evaluate the signal intensity as well as the NSR of the traditional N-sGFP and C-sGFP split-GFP system, two devices, containing split-GFP fragments and a complete or spited zinc finger protein, were built under control of a lac operon controlled T7 promoter. The complete zinc finger protein was to stimulate a PPI positive situation, while the split one was to stimulate a PPI negative situation. After overnight expressed in E.coli, Fluorescence was detected and Relative fluorescence intensity was calculated. Results showed a significantly higher signal intensity in PPI positive groups than that in the PPI negative groups. Thus validated the function of this part.
Figure 2. Evaluation of the Signal-Noise Ratio of split GFP system (A) Schematic representation of the evaluation protocol. The complete Zif-268 protein was introduced to simulate the condition where strong interaction among two proteins occur, whereas the split-zif protein was used to simulate the condition where no interaction exists. (B) Fluorescent assay showing the fluorescent intensity under two different conditions. Relative FI was calculated with normalization of the OD600 value. This experiment was run in three parallel reactions, and the data represent results obtained from at least three independent experiments. **p<0.01.
To further demonstrate the substitution system, we replaced the Zif268 region in this part into a FRB-RBS-FKBP fragment. The further experimental validation can be seen on BBa_K1997017.
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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