Difference between revisions of "Part:BBa K1997017"

Latest revision as of 01:20, 21 October 2016

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).

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_K1997017 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

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).

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K1997017 short</partinfo>
-This part is an integrated tool for protein-protein interaction research using split-GFP 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 years<sup>1 </sup>. Among which, split-GFP system, due to its wide applicability, was widely applied in various fields of researches<sup>2 </sup>.
+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 years<sup>1 </sup>. Among which, split-GFP system, due to its wide applicability, was widely applied in various fields of researches <sup>2 </sup>.
-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 application<sup>3 </sup>. To address this, we introduced a newly-developed split-GFP assay that was recently reported in 2013<sup>4 </sup> 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===
@@ Line 18: / Line 14: @@
 [[File:NUDT-017-2.jpg|500px|]]
-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.
+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_K1997017 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===
@@ Line 54: / Line 51: @@
 '''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.
+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 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.
@@ Line 64: / Line 61: @@
 ===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.
-===References===
+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.
-[1] Day, R. N. &amp; Davidson, M. W.The fluorescent protein palette: tools for cellular imaging. <i>Chem Soc Rev</i> <b>38</b>, 2887-2921, doi:10.1039/b901966a (2009).
-[2]    Pfleger, K. D.&amp; Eidne, K. A. Illuminating insights into protein-protein interactions using bioluminescence resonance           energy transfer (BRET). <i>Nature methods</i> <b>3</b>,165-174, doi:10.1038/nmeth841 (2006).
+[[File:T--NUDT_CHINA--partsfig1.jpg|700px|]]
-[3]    Kodama, Y. &amp;Hu, C. D. An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio.<i>Biotechniques</i> <b>49</b>, 793-805, doi:10.2144/000113519(2010).
+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 OD<sub>600 </sub> 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. &amp; Davidson, M. W.The fluorescent protein palette: tools for cellular imaging. <i>Chem Soc Rev</i> <b>38</b>, 2887-2921, doi:10.1039/b901966a (2009).
-[4]    Cabantous, S.<i> et al.</i> A new protein-protein interaction sensor based on tripartite split-GFP association. <i>Scientific reports</i> <b>3</b>, 2854, doi:10.1038/srep02854 (2013).
+[2] Pfleger, K. D.&amp; Eidne, K. A. Illuminating insights into protein-protein interactions using bioluminescence resonance           energy transfer (BRET). <i>Nature methods</i> <b>3</b>,165-174, doi:10.1038/nmeth841 (2006).