Difference between revisions of "Part:BBa K1997021"

Latest revision as of 01:22, 21 October 2016

P+R->sLuc-N->Zif268->sLuc-C->TER

This part is an integrated tool for protein-protein interaction research using split-LUC 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-LUC 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_K1997021 to form a brand new device expressing the proteins of GFP1-Protein1, Protein2-GFP2. The interaction between Protein1 and protein 2 could then be determined through the florescent intensity.

Sequence and Features

Sequence and Features

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 the part BBa_K1997022 using BBa_K19970022 as control.

To proof the basic function of our split-luciferase reporting system, two devices, containing split-LUC fragments and a complete (or split as control) 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.

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 and ultra-filtrated (to remove small molecules), with D-luciferin substrate solution was added into the cell lysate for the measurement of luciferase activity.

Chemo-luminescence assay showed significant variation between the PPI positive group and the PPI negative group. Thus then validated the function of this part.

Figure 2. Evaluation of the Signal-Noise Ratio of split LUC 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) Experimment showing the RLU under two different conditions. Relative RLU was calculated with normalization of RLU. 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).

[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).