Part:BBa_K4016021

HA-Trim21-PixD

This composite part is designed to generate GFP degradation with Part:BBa_K4016022 (GFPnano - Pix E) through Pix E-Pix D interaction, GFPnano’s targeting function and Trim21 based ubiquitin-proteasome degradation system.

Usage and Biology

This part only changes Pix E in Part:BBa_K4016019(click to gain more information) to Pix D. We designed this part to interact with Part:BBa_K4016022 (GFPnano - Pix E) .

Figure1. Schematic figure of BBa_K4016021 and BBa_K4016022

• 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.

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-CTAGCGTTTAAACTTAAGCTTGCCACCATGGAGTACCCCTACGACGTGCCCGACTAC-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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 204
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 873
  Illegal BamHI site found at 1411
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 161
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1251
  

Functional test

This part (HA-Trim21-Pix D) was tested together with BBa_K4016022

(GFPnano-Pix E) 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.

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