Difference between revisions of "Part:BBa K3595012"

 
 
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When knocking out gene by using CRISPR-cas9 in E.coli, there should be a series of DNA that leads the genome repairing after cutting, so that the E.coli could live after knocking out a gene.  
 
When knocking out gene by using CRISPR-cas9 in E.coli, there should be a series of DNA that leads the genome repairing after cutting, so that the E.coli could live after knocking out a gene.  
  
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===Usage and Biology===
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=Usage and Biology=
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This sequence of DNA is used to help cas9 protein to cut the specific position on the DNA. It is constructed by connecting pUC, Ampr, RBS J23119, pTarget-upArm-gRNA-downarm. It is induced by the arabinose.
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==Experimental Setup==
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*Transform the plasmid pTarget with up-arm, down-arm, and gRNA to the competent<i>E.coli DH10b </i> from company transgene.
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*Transform the plasmid pCas into the competent E.coli with previous plasmid mentioned.
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*Add arabinose
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*Cultivate the <i>E.coli </i> with the temperature at 30 degree Celsius overnight.
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*Use colony PCR to test if the gene is successfully knocked out.
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==Results==
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* As shown on the graph, the argR is knocked out with the decrease of 500bp which is the length of argR.
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[[File:T--GZ_HFI--argR.png|450px|thumb|center|Contrast of E.coli DH10b genome before and after the deletion of argR. (A)<i>DH10b </i> with argR. (B) <i>DH10b </i> genome without argR. (C) The electrophoresis graph of <i>DH10b </i> genome with and without argR. Line 1 is marker (RB-MKS); Line 2 is wildtype; Line 3 is <i>DH10b ∆argR</i> ]]
  
 
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Latest revision as of 18:42, 27 October 2020


up 500 bp DNA of E.coli genome in deleting argR gene

When knocking out gene by using CRISPR-cas9 in E.coli, there should be a series of DNA that leads the genome repairing after cutting, so that the E.coli could live after knocking out a gene.

Usage and Biology

This sequence of DNA is used to help cas9 protein to cut the specific position on the DNA. It is constructed by connecting pUC, Ampr, RBS J23119, pTarget-upArm-gRNA-downarm. It is induced by the arabinose.

Experimental Setup

  • Transform the plasmid pTarget with up-arm, down-arm, and gRNA to the competentE.coli DH10b from company transgene.
  • Transform the plasmid pCas into the competent E.coli with previous plasmid mentioned.
  • Add arabinose
  • Cultivate the E.coli with the temperature at 30 degree Celsius overnight.
  • Use colony PCR to test if the gene is successfully knocked out.

Results

  • As shown on the graph, the argR is knocked out with the decrease of 500bp which is the length of argR.
Contrast of E.coli DH10b genome before and after the deletion of argR. (A)DH10b with argR. (B) DH10b genome without argR. (C) The electrophoresis graph of DH10b genome with and without argR. Line 1 is marker (RB-MKS); Line 2 is wildtype; Line 3 is DH10b ∆argR

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]