Difference between revisions of "Part:BBa K2316001"

 
 
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<partinfo>BBa_K2316001 short</partinfo>
 
<partinfo>BBa_K2316001 short</partinfo>
  
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The parts is a triple truncation of dCas9 in HNH, Rec2, and RUVCIII2 domain with quick change to enhance dCas9 function. The truncation and quick change was based on the crystal structure of SpCas9. The protein product still functions as dCas9, which has abolished nuclease activity, but with more compact size.
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dCas9 is a catalytically inactive Cas9, which still retains it's ability to bind to DNA. For epigenetic regulation, the dCas9 is highly dependent upon the function of it's fusion partner.
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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For this part, we have deleted the HNH domain, Rec2 domain and RuvCIII-2 domain of the SP-dCas9. The triple truncation (∆REC2 ∆RuvCIII-2 ∆HNH, referred to as ‘∆3ple’) is especially of interest, since it reduces the size of the dCas9 gene to around 3.2kB which is on par with the size of Sa-Cas9. The ∆3ple truncation has slightly lower activation compared to double truncation. In order to improve the activity of ∆3ple dCas9, quick changes are further performed at various site of dCas9. Combination of quick change at Asn497Lys (QC5), Thr657Lys (QC6) and Ser1109Arg (QC15) show improved activity. It's binding capability was tested by assessing the strength in transcriptional activation via by tagging it with a VP64-p65-Rta tripartite activator.
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<html><center><img src="https://static.igem.org/mediawiki/2017/4/44/Truncation.png" >
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<p>Activation of a zsGreen reporter gene using the various truncations.</p>
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<br>
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<br>
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<img src="https://static.igem.org/mediawiki/2017/8/8f/3ple_qc_x1.png" >
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<p>Quick change mutations done on ∆3ple. QC5 and QC15 appears to give best improvement in gene activation.</p>
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<br>
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<br>
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<img src="https://static.igem.org/mediawiki/2017/5/5f/3ple_qc_x2.png" >
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<p>Combination of different mutations done on ∆3ple QC5 and QC15 as base. QC5+6 and QC15+6 appears to give best improvement in gene activation.</p>
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<br>
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<br>
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<img src="https://static.igem.org/mediawiki/2017/d/df/3ple_qc_x3.png" >
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<p>Combination of different mutations done on ∆3ple QC5+6 and QC15+6 as base.</p></html>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2316001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2316001 SequenceAndFeatures</partinfo>

Latest revision as of 16:48, 30 October 2017


∆RuvCIII-2 ∆HNH ∆REC2 Sp-dCas9 Enhanced

dCas9 is a catalytically inactive Cas9, which still retains it's ability to bind to DNA. For epigenetic regulation, the dCas9 is highly dependent upon the function of it's fusion partner.

Usage and Biology

For this part, we have deleted the HNH domain, Rec2 domain and RuvCIII-2 domain of the SP-dCas9. The triple truncation (∆REC2 ∆RuvCIII-2 ∆HNH, referred to as ‘∆3ple’) is especially of interest, since it reduces the size of the dCas9 gene to around 3.2kB which is on par with the size of Sa-Cas9. The ∆3ple truncation has slightly lower activation compared to double truncation. In order to improve the activity of ∆3ple dCas9, quick changes are further performed at various site of dCas9. Combination of quick change at Asn497Lys (QC5), Thr657Lys (QC6) and Ser1109Arg (QC15) show improved activity. It's binding capability was tested by assessing the strength in transcriptional activation via by tagging it with a VP64-p65-Rta tripartite activator.


Activation of a zsGreen reporter gene using the various truncations.



Quick change mutations done on ∆3ple. QC5 and QC15 appears to give best improvement in gene activation.



Combination of different mutations done on ∆3ple QC5 and QC15 as base. QC5+6 and QC15+6 appears to give best improvement in gene activation.



Combination of different mutations done on ∆3ple QC5+6 and QC15+6 as base.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 251
    Illegal BglII site found at 804
    Illegal BamHI site found at 1622
    Illegal XhoI site found at 2578
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1966
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2112
    Illegal BsaI site found at 2774
    Illegal BsaI.rc site found at 1027