Difference between revisions of "Part:BBa K4016024"

 
 
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<partinfo>BBa_K4016024 short</partinfo>
 
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===Usage and Biology===
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==Usage and Biology==
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Bcl-XL and LD3 are used in our project as an “OFF system”, based on their disassociation ability in response to A1155463. We replaced the PRYSPRY structure of truncated trim21 with the Bcl-xL and LD3 protein pair, and attached a GFPnano antibody to the end of Bcl-XL to achieve specific degradation of the GFP protein.
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The antibody GFP-nano is designed to bind with both Trim21 and the antigen GFP to prove that the system works well. In the design we link GFP-nano with BcL-XL, so that it can work as the bridge between target GFP and Trim21.
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<figure class="figure">
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<img src="https://static.igem.org/mediawiki/parts/3/39/T--NUDT_CHINA--Part_SchematicFigure_23-24_.png
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" class="figure-img img-fluid rounded"  height="350px">
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Figure1. Schematic figure of BBa_K4016024 and BBa_K4016023
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==Characterization==
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This part BBa_K4016024 was cloned in pXQ109 plasmid and transfected into HEK293T cell lines using Invitrogen LipofectamineTM 3000.This part is validated through 3 ways: PCR, enzyme digestion and Sequencing.
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===PCR===
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The PCR is performed with Green Taq Mix by Vazyme.
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F-Prime：5’CTAGCGTTTAAACTTAAGCTTGCCACCATGgagtctgggggag 3’
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R-Prime：5’gctgtagtccaggatTCCGTACAGTTCCACGAAGGT 3’
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The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.
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===Enzyme Digestion===
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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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===Sequecing===
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The plasmid was sequenced correct.
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K4016024 SequenceAndFeatures</partinfo>
 
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<partinfo>BBa_K4016024 parameters</partinfo>
 
<partinfo>BBa_K4016024 parameters</partinfo>
 
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===Reference===
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[1] Giordano-Attianese, G. et al. A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy. Nat Biotechnol 38, 426–432 (2020).

Latest revision as of 16:44, 21 October 2021


GFPnano-BCl-XL


Usage and Biology

Bcl-XL and LD3 are used in our project as an “OFF system”, based on their disassociation ability in response to A1155463. We replaced the PRYSPRY structure of truncated trim21 with the Bcl-xL and LD3 protein pair, and attached a GFPnano antibody to the end of Bcl-XL to achieve specific degradation of the GFP protein.

The antibody GFP-nano is designed to bind with both Trim21 and the antigen GFP to prove that the system works well. In the design we link GFP-nano with BcL-XL, so that it can work as the bridge between target GFP and Trim21.


Figure1. Schematic figure of BBa_K4016024 and BBa_K4016023


Characterization

This part BBa_K4016024 was cloned in pXQ109 plasmid and transfected into HEK293T cell lines using Invitrogen LipofectamineTM 3000.This part is validated through 3 ways: PCR, enzyme digestion and Sequencing.

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime：5’CTAGCGTTTAAACTTAAGCTTGCCACCATGgagtctgggggag 3’

R-Prime：5’gctgtagtccaggatTCCGTACAGTTCCACGAAGGT 3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.


Enzyme Digestion

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.

Sequecing

The plasmid 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 35





Reference

[1] Giordano-Attianese, G. et al. A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy. Nat Biotechnol 38, 426–432 (2020).