Part:BBa_K1997012
P+R->sHRP-N->Zif268-> sHRP-C->TER
This part is an integrated tool for protein-protein interaction research using split-HRP system as reporter. the "Zif268" 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 researches2 .
However, researches showed that previous split-GFP based sensors always suffer from poor folding and/or self-assembly background fluorescence, thus severely limited their further application3 . To address this, we introduced a newly-developed split-GFP assay that was recently reported in 20134 into iGEM registry. This assay was based on tripartite association between two 20 amino-acids long GFP tags, GFP10 and GFP11 respectively, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP.
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-HRP fragment at the same time using Golden-Gate Assembly, was introduced to dramatically simplify the cloning process).
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_K1997012 to form a brand new device expressing the proteins of s-HRP-N-Protein1, Protein2-s-HRP-C. The interaction between Protein1 and protein 2 could then be determined through the absorbance at 450 nm.
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 1171
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1174
Illegal BsaI.rc site found at 907
Illegal SapI.rc site found at 1288
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
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).
[3] Kodama, Y. &Hu, C. D. An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio.Biotechniques 49, 793-805, doi:10.2144/000113519(2010).
[4] Cabantous, S. et al. A new protein-protein interaction sensor based on tripartite split-GFP association. Scientific reports 3, 2854, doi:10.1038/srep02854 (2013).
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