Difference between revisions of "Part:BBa K4016020"

Revision as of 14:42, 20 October 2021

GFPnano-PixD

This composite part is designed to generate GFP degradation with Part:BBa_K4016019 (HA-Trim21-PixE) through Pix E-Pix D interaction, GFPnano’s targeting function and Trim21 based ubiquitin-proteasome degradation system.

Usage and Biology

The antibody GFP-nano is designed to bind with both Trim21 and the antigen GFP to prove that the system really works. In the design we link GFP-nano with hIgG1-Fc, so that it can work as the bridge between target GFP and Trim21

Pix D and Pix E, can associate in the dark and dissociate on blue light stimulation. 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. [1]~[5]

This composite part is designed to interact with BBa_K4016019 (HA-Trim21-PixE), to test the if the Pix D/Pix E’s dissociation in light can inhabit the degradation rate of GFP by Trim21, in order to design an “OFF switch” in our project.

• 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 was validated through four ways:PCR, enzyme digestion, sequencing and functional test.

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime：5’CTAGCGTTTAAACTTAAGCTTGCCACCATGgagtctgggggag 3’

R-Prime：5’TGGATATCTGCAGAATTCTTAttagaggtcgaggaaaaagttatc 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

Functional test

This part (GFPnano-PixD) was tested together with BBa_K4016019 (HA-Trim21-PixE) and using they two respectively as control. (using eGFP~ Fluc-Rluc stable expressing 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.

• 2.Dual luciferase assay

(1)Wash HEK293T cells in 6-well plate with PBS and trypsinize prior to resuspension in fresh complete medium in a 15 ml microcentrifuge tube.

(2)Dispense 100ul of cell suspension (approximately 30000 cells per well) into 96 well plates.

(3)Capture the fluorescent image before apply blue light.(24h after transfection)

(4)Apply the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48/72 h before sampling and analysis assay. Capture the fluorescent image at 48/72 h respectively

(5)At 96h , add Reporter cell lysates in 96 well plates. (100uL per well)

(6)After extensive lysis, centrifugation at 10000-15000g for 3-5 min. Take the supernatant for assay.

(7)Thaw firefly luciferase assay reagent and Renilla luciferase assay buffer, and bring to room temperature. Renilla luciferase assay substrate (100x) was placed on an ice bath or on an ice box for later use.

(8)Prepare Renilla luciferase assay working solution by adding Renilla luciferase assay substrate (100x) at 1:100 in an amount of 100 µ l per sample.

(9)Switch on the microplate reader, set the assay interval to 2 s and the assay time to 10 s.

(10)Take 20 to 100 ul of each sample for assay

(11)Add 100 ul of firefly luciferase assay reagent, measure the RLU (relative light unit) after mixing. Reporter cell lysate was used as a blank control.

(12)Add 100 ul of Renilla luciferase assay working solution

(13)The RLU value obtained from the Fluc assay was divided by the RLU value obtained from the Rluc assay. The degree of reporter gene activation of interest was compared between different samples according to the ratio obtained.

Result

Figure 2 Characterization of GFPnano - PixD/HA-Trim21-PixE interaction.Dual luciferase system was used and Rluc act as Internal reference. Via Fluc/Rluc ratio to show the amount of degradation.

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 3 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