Difference between revisions of "Part:BBa K4016000"
 
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==Usage and Biology==
 
==Usage and Biology==
  
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PixE (Slr1693) is a PatA-like two-component response regulator that interacts with PixD in vivo.
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PixE (Slr1693) is a PatA-like two-component response regulator that interacts with PixD in vivo.[1]
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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.  
+
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.
 
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.
  
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<html>
  
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==Characterization==
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<figure class="figure">
 +
<img src="https://static.igem.org/mediawiki/parts/1/17/T--NUDT_CHINA--Part_PixD-PixE_Schematic.png" class="figure-img img-fluid rounded"  height="250px">
  
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===Sequencing===
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</figure>
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This part is sequenced as correct after construction.
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</html>
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Figure1. Schematic figure of PixE-PixD interaction under blue light stimulation
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 +
 +
 +
==Characterization==
 +
This part was measured through 3 ways:PCR, enzyme digestion, sequencing.
  
 
===PCR===
 
===PCR===
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The PCR is performed with Premix EX Taq.
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The PCR is performed with Green Taq.
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F-Prime：5’GGAGGATCTGGTGGTatgagcaattcagttttgtcc 3’（oXQ212 forward prime）
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F-Prime：5’GGAGGATCTGGTGGTatgagcaattcagttttgtcc 3’
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R-Prime：5’TGGATATCTGCAGAATTCTTAtcaggagttggttttattggtg 3’（oXQ213 reverse prime）
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R-Prime：5’TGGATATCTGCAGAATTCTTAtcaggagttggttttattggtg 3’
 
The PCR protocol is selected based on the Users Manuel.  The Electrophoresis was performed on a 1％ Agarose glu.  
 
The PCR protocol is selected based on the Users Manuel.  The Electrophoresis was performed on a 1％ Agarose glu.  
  
  
 
===Enzyme digestion test===
 
===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 cutting procedure was performed with Hind III EcoR I restriction endonuclease bought.
 
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.
+
The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose gel.
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−
 
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<partinfo>BBa_K4016000 parameters</partinfo>
 
<partinfo>BBa_K4016000 parameters</partinfo>
 
<!-- -->
 
<!-- -->
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==Experimental Validation==
 +
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===
 +
<html>
 +
 +
<figure class="figure">
 +
<img src="https://static.igem.org/mediawiki/parts/6/65/T--NUDT_CHINA--Part_Validation_SEAP_PixE-PixD.png
 +
" class="figure-img img-fluid rounded"  height="350px">
 +
 +
</figure>
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 +
</html>
 +
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===
 +
<html>
 +
 +
<figure class="figure">
 +
<img src="https://2021.igem.org/wiki/images/d/d6/T--NUDT_CHINA--Part_Result_00-01_.png
 +
" class="figure-img img-fluid rounded"  height="350px">
 +
 +
</figure>
 +
 +
</html>
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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.
 +
 +
  
  

Latest revision as of 04:38, 21 October 2021


PixE

PixE is a PatA-like two-component response regulator that interacts with PixD in vivo. It control the phototaxis in the cyanobacterium Synechocystis sp.


Usage and Biology

PixE (Slr1693) is a PatA-like two-component response regulator that interacts with PixD in vivo.[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 measured through 3 ways:PCR, enzyme digestion, sequencing.

PCR

The PCR is performed with Green Taq. F-Prime：5’GGAGGATCTGGTGGTatgagcaattcagttttgtcc 3’ R-Prime：5’TGGATATCTGCAGAATTCTTAtcaggagttggttttattggtg 3’ The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu.


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.


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]



Experimental Validation

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.