Difference between revisions of "Part:BBa K1997014"
Zhuchushu13 (Talk | contribs) |
Zhuchushu13 (Talk | contribs) |
||
| Line 4: | Line 4: | ||
This part is an integrated tool for protein-protein interaction research using split-GFP system as reporter. | This part is an integrated tool for protein-protein interaction research using split-GFP system as reporter. | ||
| − | + | ||
===Usage and Biology=== | ===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>. | ||
| + | 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. | ||
| + | |||
| + | [[File:sg10g11g1-9figi.jpg]] | ||
| + | |||
| + | ===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 (Figure 2), 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 3). 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. | ||
| + | |||
| + | |||
| + | ===Sequence and Features=== | ||
<!-- --> | <!-- --> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1997014 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1997014 SequenceAndFeatures</partinfo> | ||
| + | ==Experimental Validation== | ||
| − | + | This part is validated through four ways: enzyme cutting, PCR, Sequence, and functional testing | |
| − | ===Functional | + | |
| − | + | ===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. | ||
| + | |||
| + | [[File:NUDT-014-1.jpg|300px|]] | ||
| + | |||
| + | ===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=== | ||
| + | |||
| + | |||
| + | ===References=== | ||
| + | |||
| + | [1] Drewes G, Bouwmeester T.Global approaches to protein – protein interaction[ J ]. Curr Opin Cell Biol, 2003, 15(2): 199-205. | ||
| + | |||
| + | [2] Collura V, Boissy G. From protein -protein complexes to interactomics [ J ]. Subcell Biochem, 2007, 43: 135-83. | ||
| + | |||
| + | [3] M isteli T, Spector DL. Application of the green fluorescent protein in cell biology and biotechnology[ J ]. Nat Biotechnol, 1997, 15( 10): 961 - 964. | ||
| + | |||
| + | [4] Baird G. S, Zacharias DA, Tsien RY. Circular permutation and receptor insertion within green fluorescent proteins[ J ]. Proc Natl Acad Sci USA , 1999, 96( 20): 11241-11246. | ||
| + | |||
| + | [5] Ghosh I, Hamilton AD, Regan L. Antiparallel leucinezipper –directed protein reassembly: application to the green fluorescent protein[ J ]. J Am Chem Soc, 2000, 122: 5658-5659. | ||
Revision as of 17:06, 17 October 2016
GFP10->Zif268->GFP11->RBS->GFP1-9
This part is an integrated tool for protein-protein interaction research using split-GFP system as reporter.
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 (Figure 2), 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 3). 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.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 51
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 208
Illegal AgeI site found at 292 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 380
Illegal BsaI.rc site found at 112
Illegal BsaI.rc site found at 443
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
References
[1] Drewes G, Bouwmeester T.Global approaches to protein – protein interaction[ J ]. Curr Opin Cell Biol, 2003, 15(2): 199-205.
[2] Collura V, Boissy G. From protein -protein complexes to interactomics [ J ]. Subcell Biochem, 2007, 43: 135-83.
[3] M isteli T, Spector DL. Application of the green fluorescent protein in cell biology and biotechnology[ J ]. Nat Biotechnol, 1997, 15( 10): 961 - 964.
[4] Baird G. S, Zacharias DA, Tsien RY. Circular permutation and receptor insertion within green fluorescent proteins[ J ]. Proc Natl Acad Sci USA , 1999, 96( 20): 11241-11246.
[5] Ghosh I, Hamilton AD, Regan L. Antiparallel leucinezipper –directed protein reassembly: application to the green fluorescent protein[ J ]. J Am Chem Soc, 2000, 122: 5658-5659.