Part:BBa_K1997005
sHRP-N->FRB->RBS->FKBP-> sHRP-C
This part is an integrated tool for protein-protein interaction research using split-HRP system as reporter. the "FRB-RBS-FKBP" subpart can be easily replaced using Golden Gate technique with BsaI
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 154
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 396
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1324
Illegal BsaI.rc site found at 679
Illegal SapI.rc site found at 1438
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
The function of this part was validated together with BBa_K1997005, since it was a composite part putting this BBa_K1997005 under the control of lac promoter.
The basic function of our split-HRP reporting system has been determined in the function validation of BBa_K1997013 and BBa_K1997012. Building on that, BBa_K1997011 was built to demonstrate the imbedded substitution system. For such matters, the Zif268 region in BBa_K1997015 was replaced into a “FRB-RBS-FKBP” fragment using Golden Gate Assembly. The cloning results were validated through sequencing.
For functional 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 and ultra-filtrated (to remove small molecules), 0.4nM of Rapamycin was added together with TMB substrate solution (with heme supplementation) into the cell lysate for the induction of protein-protein interaction and 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 Rapamycin positive group and the Rapamycin negative group. Thus then validated the function of this part.
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).
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