Part:BBa_K1997017

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

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 researches ² .

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).

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_K1997004 to form a brand new

device expressing the proteins of sGFP-N-Protein1, Protein2-sGFP-C and. 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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1381
  Illegal BsaI.rc site found at 736
  Illegal BsaI.rc site found at 1595
  

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

Building on the results of BBa_K1997015 and BBa_K1997016, further experiments

were conducted to demonstrate the imbedded substitution system. For such

matters, BBa_K1997017 was constructed by replacing the Zif268 region in

BBa_K1997015 into a “FRB-RBS-FKBP” fragment. The cloning results were

validated through sequencing.

For function 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, 0.4nM of Rapamycin was added into the cell

lysate to induce the protein-protein reaction.

Fluorescence intensity measured right after the addition of Rapamycin showed a

significant improvement on relative FI, thus validated the function of this

part.

Figure 1. Rapamycin-induced sGFP-N-FRB/sGFP-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-GFP fragments and reconstruct its structure for

fluorescence generation. (B) Fluorescent assay showing the fluorescent

intensity with/without Rapamycin induction. Relative FI was calculated with

normalization of the OD600 value. For Fold change Relative FI, relative FI of

the group without Rapamycin induction was set arbitrarily as 1.0, and the

levels of the other groups were adjusted correspondingly. The concentration of

Rapamycin used in the experiment was 40nM. 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).