Difference between revisions of "Part:BBa K4016001"

(→‎PCR)
Line 32: Line 32:
 
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 glu.
 +
  
  
Line 43: Line 44:
 
<partinfo>BBa_K4016001 parameters</partinfo>
 
<partinfo>BBa_K4016001 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
===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.

Revision as of 15:51, 16 October 2021


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. 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.


Characterization

Sequencing

This part is sequenced as correct after construction.

PCR

Methods The PCR is performed with Premix EX Taq.

F-Prime：5’GGCGGCGGCAGCGGCatgagtttgtaccgtttgatttac 3’（oXQ214 forward prime）

R-Prime：5’TGGATATCTGCAGAATTCTTAttagaggtcgaggaaaaagttatc 3’（oXQ215 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.