Difference between revisions of "Part:BBa K3044016"
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<partinfo>BBa_K3044023 short</partinfo>
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The assembled sgRNA-dCas9 system can be used for silencing of GFP (<partinfo>BBa_E0040</partinfo>) expression. This system is made transferable between bacteria by the implementation of OriT(<partinfo>BBa_J01003</partinfo>) in the plasmid.
 
The assembled sgRNA-dCas9 system can be used for silencing of GFP (<partinfo>BBa_E0040</partinfo>) expression. This system is made transferable between bacteria by the implementation of OriT(<partinfo>BBa_J01003</partinfo>) in the plasmid.
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dCas9 is the inactive form of the endonuclease Cas9. In contrast to Cas9, dCas9 does not cleave the DNA. Instead, it sterically blocks the elongation of transcription as a result of the sgRNA-DNA interaction. This will only repress the expression of the target gene thus avoiding destruction of the gene. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/]
 
dCas9 is the inactive form of the endonuclease Cas9. In contrast to Cas9, dCas9 does not cleave the DNA. Instead, it sterically blocks the elongation of transcription as a result of the sgRNA-DNA interaction. This will only repress the expression of the target gene thus avoiding destruction of the gene. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/]
  
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This sgRNA sequence targets residue 325-245 on the template strand in the GFP sequence and have an efficiency of 67.06. When forming a complex with the dCas9, the sgRNA guides the dCas9 to the appropriate target gene. The sgRNA is designed according to the location of the PAM sequence in the target gene, because the PAM sequence is recognized by and activates the dCas9. The sgRNA consists of 100 nucleotides of which 20 nucleotides base pair with the target DNA sequence and the rest is called a dCas9 handle and interacts with the dCas9 protein.
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This sgRNA sequence targets residue 75-95 on the template strand in the GFP sequence and have an efficiency of 59.09. When forming a complex with the dCas9, the sgRNA guides the dCas9 to the appropriate target gene. The sgRNA is designed according to the location of the PAM sequence in the target gene, because the PAM sequence is recognized by and activates the dCas9. The sgRNA consists of 100 nucleotides of which 20 nucleotides base pair with the target DNA sequence and the rest is called a dCas9 handle and interacts with the dCas9 protein.
  
 
The plasmid is made conjugative by the insertion of OriT (<partinfo>BBa_J01003</partinfo>). This makes the sgRNA-dCas9 transferable between bacteria.  
 
The plasmid is made conjugative by the insertion of OriT (<partinfo>BBa_J01003</partinfo>). This makes the sgRNA-dCas9 transferable between bacteria.  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3044016 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3044023 parameters</partinfo>
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<partinfo>BBa_K3044016 parameters</partinfo>
 
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Revision as of 09:33, 14 October 2019


Conjugative sgRNA-dCas9 system

The assembled sgRNA-dCas9 system can be used for silencing of GFP (BBa_E0040) expression. This system is made transferable between bacteria by the implementation of OriT(BBa_J01003) in the plasmid.

This dCas9 is a codon optimized version of BBa_K1150000 for expression in E. coli. It forms a complex with the single guide RNA (sgRNA), and together this system can be used for silencing of specific target genes. dCas9 is the inactive form of the endonuclease Cas9. In contrast to Cas9, dCas9 does not cleave the DNA. Instead, it sterically blocks the elongation of transcription as a result of the sgRNA-DNA interaction. This will only repress the expression of the target gene thus avoiding destruction of the gene. [1]

This sgRNA sequence targets residue 75-95 on the template strand in the GFP sequence and have an efficiency of 59.09. When forming a complex with the dCas9, the sgRNA guides the dCas9 to the appropriate target gene. The sgRNA is designed according to the location of the PAM sequence in the target gene, because the PAM sequence is recognized by and activates the dCas9. The sgRNA consists of 100 nucleotides of which 20 nucleotides base pair with the target DNA sequence and the rest is called a dCas9 handle and interacts with the dCas9 protein.

The plasmid is made conjugative by the insertion of OriT (BBa_J01003). This makes the sgRNA-dCas9 transferable between bacteria.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 478
    Illegal BglII site found at 1552
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4547
    Illegal NgoMIV site found at 4557
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 4449