Coding

Part:BBa_K4016000

Designed by: Zhixin Fang   Group: iGEM21_NUDT_CHINA   (2021-10-01)
Revision as of 12:46, 20 October 2021 by ZhixinFang (Talk | contribs)


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. 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. 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.

T--NUDT_CHINA--Part_SchematicFigure_tryyyy.png

Characterization

Sequencing

This part is sequenced as correct after construction.


PCR

The PCR is performed with Premix EX Taq. F-Prime：5’GGAGGATCTGGTGGTatgagcaattcagttttgtcc 3’（oXQ212 forward prime） R-Prime：5’TGGATATCTGCAGAATTCTTAtcaggagttggttttattggtg 3’（oXQ213 reverse prime） The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu.


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. The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.



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]



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.

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Parameters
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