Difference between revisions of "Part:BBa K1997023"

Revision as of 01:06, 21 October 2016

P+R->sLuc-N->FRB->RBS->FKBP->sLuc-C->TER

This part is an integrated tool for protein-protein interaction research using split-LUC system as reporter. the "FRB-RBS-FKBP" 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-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_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

TBuilding on the results of BBa_K1997021 and BBa_K1997022, further experiments were conducted to demonstrate the imbedded substitution system. For such matters, BBa_K1997020 was constructed by replacing the Zif268 region in BBa_K1997023 into a “FRB-RBS-FKBP” fragment. The cloning results were validated through sequencing.

For functional validation, we used Rapamycin to induce the interaction between FRB and FKBP. 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), 0.4nM of Rapamycin was added together with D-luciferin substrate solution into the cell lysate for the induction of protein-protein interaction and measurement of luciferase activity.

Figure 2. Rapamycin-induced sLUC-N-FRB/sLUC-C-FKBP interaction. (A) Schematic representation of the rapamycin induced protein-protein interaction. The adding of rapamycin would induce the interaction between FRB and FKBP, thus shortened the range between split-LUC fragments and reconstruct its structure for fluorescence generation. (B)Experimment showing the RLU with/without Rapamycin induction. This experiment was run in three parallel reactions, and the data represent results obtained from at least three independent experiments. **p<0.01.

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

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).