Difference between revisions of "Part:BBa K4016000"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4016000 short</partinfo>
 
<partinfo>BBa_K4016000 short</partinfo>
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PixE is a PatA-like two-component response regulator that interacts with PixDin vivo. It control the phototaxis in the cyanobacterium Synechocystis sp
 
PixE is a PatA-like two-component response regulator that interacts with PixDin vivo. It control the phototaxis in the cyanobacterium Synechocystis sp
  
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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.  
 
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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===Experimental Validation===
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==Sequencing==
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This part is sequenced as correct after construction.
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==PCR==
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Methods
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The PCR is performed with Premix EX Taq.
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F-Prime：5’GGAGGATCTGGTGGTatgagcaattcagttttgtcc 3’（oXQ212 forward prime）
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R-Prime：5’TGGATATCTGCAGAATTCTTAtcaggagttggttttattggtg 3’（oXQ213 reverse prime）
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The PCR protocol is selected based on the Users Manuel.  The Electrophoresis was performed on a 1％ Agarose glu. The result of the agarose electrophoresis was shown on the picture below.
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==Enzyme digestion test==
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Methods
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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.
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The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.
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Revision as of 03:21, 15 October 2021


PixE

PixE is a PatA-like two-component response regulator that interacts with PixDin 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.

Experimental Validation

Sequencing

This part is sequenced as correct after construction.

PCR

Methods 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. The result of the agarose electrophoresis was shown on the picture below.

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]