Part:BBa_K4016019
HA-Trim21-PixE
This composite part is designed to generate GFP degradation with Part:BBa_K4016020 (GFPnano - Pix D) through Pix E-Pix D interaction, GFPnano’s targeting function and Trim21 based ubiquitin-proteasome degradation system.
Usage and Biology
Protein-protein interactions are powerful tools for manipulating biological processes, and opto-genetically - mediated induction of protein interactions has high spatial resolution without the need for exogenous cofactors. In this case, the dimer ”Pix D- Pix E” is introduced in our project in order to reach such function.
The two protein, Pix D and Pix E, can associate in the dark into large multi-subunit complexes that dissociate into dimers of Pix D and monomers of Pix E within seconds upon blue light stimulation. [1]~[5] Fusing Pix D/Pix E fragment to the Trim21 and its targeting module, we can realize the Trim21-induced degradation on it’s target protein in the dark, and the Pix D-Pix E dissociation on blue light can induced the dissociation of Trim21 and its targeting module, so as to stop the degradation.
In our project, our composite part BBa_K4016019 is interacting with Part:BBa_K4016020 (GFPnano - Pix D ), to specifically recognize and degrade GFP in the dark and stop the degradation in the light. If this can work, we can make an “OFF switch” in a system mediated by blue light.
- Here is the mechanism of the system:
In the dark environment:
1.HA-Trim21-PixE connect with PixD-GFPnano through PixE-PixD interaction and forms a complex
2.GFPnano specifically recognize eGFP
3.eGFP is degraded by ubiquitin-proteasome system recruited by Trim21
In the blue light:
1. PixD-PixE dissociate
2. The degradation of eGFP stop because of the lack of trim21.
Figure1. Schematic figure of BBa_K4016019 and BBa_K4016020
Characterization
This part is validated through four ways: enzyme cutting, PCR, Sequence, and functional testing.
PCR
The PCR is performed with Green Taq Mix by Vazyme.
F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGGAGGTTCAACTGGAGGAGTCTG-3’
R-Prime: 5’- TGGATATCTGCAGAATTCTTACACGTTCACGCCGCCGTCGAT-3’
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.
Enzyme Digestion
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 cutting procedure was performed with Hind III EcoR I restriction endonuclease bought. The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.
Sequecing
The plasmid was sequenced correct.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 204
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 873
Illegal BamHI site found at 1411 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 161
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1251
Functional test
This part (HA-Trim21-PixE) was tested together with BBa_K4016020 (GFPnano-PixD) and using they two respectively as control. (using eGFP~ Fluc-Rluc stable expressing HK293T cell line as follow, Rluc act as Internal reference)
Method
- 1.Cell transfection
(1)Seed HEK293T cells into 6-well cell culture plates.
(2)Culture for 16 h before transfection
(3)Total plasmid mixes of 800ng per well are mixed thoroughly in DMEM before a polyethylenimine (PEI) solution (1 mg/ml) is added into the plasmid mixture in a ratio of 1:5 (plasmid weight/PEI weight)
(4)The plasmid–PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for at least 6 h.
(5)Cells are then changed into fresh medium and culture for 18 h before subculture.
Result
Figure 2 Characterization of GFPnano - PixD/HA-Trim21-PixE interaction. Dual luciferase
The result shows a very small fluctuation change over the control group, which reply a little amount of EGFP~Fluc’s degradation. The interaction of PixD and PixE is not as ideal as we thought.
We tried to use another promoter EF1a instead of CMV to see is it the error of promoter CMV that make the system not expressed, so as to gain such a negative result.
Figure 5 Characterization of GFPnano - PixD/HA-Trim21-PixE interaction after change the promoter to EF1a.
The new promoter EF1a still showed a negative result, in which the rate of Fluc degradation is slight. The reason of this might be the mechanism of PixD/PixE interaction. PixE and PixD associate in the dark into large multi-subunit complexes, this complex may be hard to drive the Trim21 to the target protein or itself has other unknown functions that inhibit this process.
Reference
[1] Dine E, Gil AA, Uribe G, Brangwynne CP, Toettcher JE. Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli. Cell Syst. 2018 Jun 27;6(6):655-663.e5. doi: 10.1016/j.cels.2018.05.002. Epub 2018 May 30. PMID: 29859829; PMCID: PMC6023
[2] Yuan H, Bauer CE. PixE promotes dark oligomerization of the BLUF photoreceptor PixD. Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11715-9. doi: 10.1073/pnas.0802149105. Epub 2008 Aug 11. PMID: 18695243; PMCID: PMC2575306.
[3] Masuda S, Hasegawa K, Ishii A, Ono TA. Light-induced structural changes in a putative blue-light receptor with a novel FAD binding fold sensor of blue-light using FAD (BLUF); Slr1694 of synechocystis sp. PCC6803. Biochemistry. 2004 May 11;43(18):5304-13. d
[4] Okajima K, Yoshihara S, Fukushima Y, Geng X, Katayama M, Higashi S, Watanabe M, Sato S, Tabata S, Shibata Y, Itoh S, Ikeuchi M. Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria. J Biochem. 200
[5] Sugimoto Y, Masuda S. In vivo localization and oligomerization of PixD and PixE for controlling phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. J Gen Appl Microbiol. 2021 Jun 3;67(2):54-58. doi: 10.2323/jgam.2020.06.001. Epub 2020 Dec 21. PMID
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