Part:BBa_K1997004
sGFP-N->Zif268->sGFP-C
This part is an integrated tool for protein-protein interaction research using split-GFP system as reporter. the "Zif268" subpart can be easily replaced using Golden Gate technique with BsaI
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 604
Illegal AgeI site found at 688
Illegal AgeI site found at 772 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 775
Illegal BsaI.rc site found at 508
Illegal BsaI.rc site found at 989
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 K1997016 and 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.
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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