Difference between revisions of "Part:BBa K1997013"
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===Usage and Biology===
 
===Usage and Biology===
  
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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>.
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Intercellular protein–protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection, and synaptic transmission, but they are challenging to visualize because existing techniques have insufficient sensitivity and/or specificity.<sup>1–3 </sup>
  
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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.
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However, a split horseradish peroxidase (sHRP) has been reported as a sensitive and specific tool for the detection of intercellular PPIs.
  
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===Special Design===
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The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft<sup>4 </sup>, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable to studying mechanisms of communication between a variety of cell types.
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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-HRP fragment at the same time using Golden-Gate Assembly, was introduced to dramatically simplify the cloning process).
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[[File:sHRPfig1.jpg|500px|]]
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[[File:013.jpg|500px|]]
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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_K1997012 to form a brand new device expressing the proteins of s-HRP-N-Protein1, Protein2-s-HRP-C. The interaction between Protein1 and protein 2 could then be determined through the absorbance at 450 nm.
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===Sequence and Features===
 
===Sequence and Features===
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The result of the agarose electrophoresis was shown on the picture below.
 
The result of the agarose electrophoresis was shown on the picture below.
  
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[[File:NUDT-013-1.jpg|300px|]]
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[[File:NUDT-013-1.jpeg|300px|]]
  
 
===Enzyme digestion test ===
 
===Enzyme digestion test ===
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===Functional Test===
 
===Functional Test===
  
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<partinfo>BBa_K1997013 parameters</partinfo>
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This part was tested together with K1997012 using K1997013 as control.
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To proof the basic function of our split-HRP reporting system, two devices, containing split-HRP fragments and a complete or split 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. 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), TMB substrate solution (with heme supplementation) were added into the cell lysate for the measurement of HRP activity. The plate was then incubated under 37°C for 5min before adding the Stop solution.
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Value of OD<sub>450 </sub> obtained after adding the Stop Solution showed significant variation between the PPI positive group and the PPI negative group. Thus then validated the function of this part.
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[[File:T--NUDT_CHINA--partsfig4.jpg|700px|]]
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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_K1997011. 
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===References===
 
===References===
  
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[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).
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1. Kim, S.A., Tai, C.-Y., Mok, L.-P., Mosser, E.A. & Schuman, E.M. Calcium-dependent
 +
dynamics of cadherin interactions at cell-cell junctions. Proc. Natl. Acad. Sci. USA
 +
108, 9857–9862 (2011).
  
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[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).
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2. Feinberg, E.H. et al. GFP Reconstitution Across Synaptic Partners (GRASP) defnes
 +
cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008).
  
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[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).
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3. Liu, D.S., Loh, K.H., Lam, S.S., White, K.A. & Ting, A.Y. Imaging trans-cellular neurexinneuroligin interactions by enzymatic probe ligation. PLoS One 8, e52823 (2013).
  
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[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).
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4. Craig, A.M. & Kang, Y. Neurexin-neuroligin signaling in synapse development. Curr.
 +
Opin. Neurobiol. 17, 43–52 (2007).

Revision as of 18:55, 19 October 2016


P+R->sHRP-N->Zif-N->RBS->Zif-C->sHRP-C->TER

This part is an integrated tool for protein-protein interaction research using split-HRP system as reporter. the "Zif-N->RBS->Zif-C" subpart can be easily replaced using Golden Gate technique with BsaI

Usage and Biology

Intercellular protein–protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection, and synaptic transmission, but they are challenging to visualize because existing techniques have insufficient sensitivity and/or specificity.1–3

However, a split horseradish peroxidase (sHRP) has been reported as a sensitive and specific tool for the detection of intercellular PPIs.

The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft4 , enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable to studying mechanisms of communication between a variety of cell types.

Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 382
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 624
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1003
    Illegal AgeI site found at 1087
    Illegal AgeI site found at 1207
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1210
    Illegal BsaI.rc site found at 907
    Illegal SapI.rc site found at 1324

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.

File:NUDT-013-1.jpeg

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 K1997012 using K1997013 as control.

To proof the basic function of our split-HRP reporting system, two devices, containing split-HRP fragments and a complete or split 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. 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), TMB substrate solution (with heme supplementation) were added into the cell lysate for the measurement of HRP activity. The plate was then incubated under 37°C for 5min before adding the Stop solution.

Value of OD450 obtained after adding the Stop Solution showed significant variation between the PPI positive group and the PPI negative group. Thus then validated the function of this part.

T--NUDT CHINA--partsfig4.jpg

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_K1997011.


References

1. Kim, S.A., Tai, C.-Y., Mok, L.-P., Mosser, E.A. & Schuman, E.M. Calcium-dependent dynamics of cadherin interactions at cell-cell junctions. Proc. Natl. Acad. Sci. USA 108, 9857–9862 (2011).

2. Feinberg, E.H. et al. GFP Reconstitution Across Synaptic Partners (GRASP) defnes cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008).

3. Liu, D.S., Loh, K.H., Lam, S.S., White, K.A. & Ting, A.Y. Imaging trans-cellular neurexinneuroligin interactions by enzymatic probe ligation. PLoS One 8, e52823 (2013).

4. Craig, A.M. & Kang, Y. Neurexin-neuroligin signaling in synapse development. Curr. Opin. Neurobiol. 17, 43–52 (2007).