Part:BBa_K4016001

PixD

PixD is a blue-light-using-flavin (BLUF)-type photoreceptor which was shown to elicit critical roles for controlling phototaxis.

Usage and Biology

PixD is a ~17 kDa protein that has the blue-light-using-flavin (BLUF) domain. The BLUF domain binds a flavin as a chromophore, and it induces structural changes when it is excited by blue light.[1]

PixD and PixE can associate in the dark into large multi-subunit complexes that dissociate into dimers of PixD and monomers of PixE within seconds upon blue light stimulation. Upon a shift back to darkness, PixD cycles back to its binding-competent state within seconds to re-form complexes. [2]

In our project, this part was used to build light-inducible targeting module for the Trim21, in order to make target protein degradation mediated by PixE/PixD interaction in blue light/dark.

Figure1. Schematic figure of PixE-PixD interaction under blue light stimulation

Characterization

This part was validated through 3 ways:PCR, enzyme digestion and sequencing.

PCR

The PCR is performed with Green Taq.

F-Prime：5’GGCGGCGGCAGCGGCatgagtttgtaccgtttgatttac 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 test

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 gel.

Sequencing

This part was sequenced correct.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional test

We constructed tetR-PixE PixD-Vp64 and TCE-SEAP to test the expression of this part. VP64 is a transcriptional activator. When fused to another protein domain that can bind near the promoter of a gene, VP64 acts as a strong transcriptional activator. TetR can recognize and combine with TCE then inhibit its downstream transcription. The interaction of PixD and PixE would restrain the TCE’s inhibition and start the transcription of SEAP.

Method

1. Cell transfection and stimulation:

(a) Seed approximately 5×104 cells into 24-well cell culture plates.

(b) Culture for 16 h before transfection.

(c) Total plasmid mixes of 500 ng per well are mixed thoroughly in serum-free DMEM before a polyethylenimine (PEI) solution (1 mg/mL) is added into the plasmid mixture in a ratio of 1:3 (plasmid weight/PEI weight).

(d) 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.

(e) Cells are then changed into fresh medium and applied with 5mA blue light(dark/blue 2/28S) for before sampling and analysis assay.

2. SEAP assay in vitro.

(a) Sample 200μL culture medium from each well, heat inactivate at 65℃ for 30 min.

(b) During the heat inactivation procedure, warm up 2×SEAP buffer (100μL/well) at 37℃.

(c) Add 1/5 buffer volume of pNPP (20μL/well) substrate into the 2×buffer to prepare the “Detection Mixture.”

(d) Add 80μL heated medium into the 96-well plate, add 120μL Detection Mixture.

(e) Measure absorption at 405 nm, 30 s per read for 10 reads.

(f) Calculate enzymatic activity.

Result

Figure 2 Result of SEAP test. The SEAP activity was calculated at 24h and 48h after transfection.

The cells showed a nearly 40% decrease in SEAP activity after 24h blue light exposure, compared with the group exposed to dark. And after 48h, the SEAP activity in blue light group was reduce to 0. This can validate PixD and PixE’s interaction.

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.