Difference between revisions of "Part:BBa K1997004"

Revision as of 08:12, 20 October 2016

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

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.

Figure 1. 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 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_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).