Part:BBa_K1997013

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.

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.

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).