Part:BBa_K1997019

P+R->GFP10->Zif-N->RBS->Zif-C->GFP11->RBS->GFP1-9->TER

This part is an integrated tool for protein-protein interaction research using split-GFP system as reporter.

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.

Figure 1. Schematic representation of different approaches for split-GFP. Shown also were the method we stimulate the PPI in our experiments.

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_K1997014 to form a brand new device expressing the proteins of GFP10-Protein1, Protein2-GFP11 and GFP1-9. The interaction between Protein1 and protein 2 could then be determined through the green florescent intensity.

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 279
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 436
  Illegal AgeI site found at 520
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 644
  Illegal BsaI.rc site found at 340
  Illegal BsaI.rc site found at 707
  

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 BBa_K1997018 using BBa_K1997019 as control. After overnight expressed in E.coli, relative fluorescence intensity was calculated.

Figure 3. 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 ₆₀₀ 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 evaluate the signal intensity as well as the NSR of our newly-introduced 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. Similar devices built with traditional N-sGFP and C-sGFP approach (registered as BBa_K1997015 and BBa_K1997016) were assigned as control. After overnight expressed in E.coli, Relative fluorescence intensity was calculated and higher signal intensity and NSR was shown on the newly-introduced system.

Figure 4. Evaluation of the Signal-Noise Ratio of split GFP system (A) Schematic representation of different approaches for split-GFP. 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_K1997020.

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).