Difference between revisions of "Team:NTU SINGAPORE/Basic Part"

 
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     <pstyle="text-align:center"> Functional units of DNA.</p>
 
     <pstyle="text-align:center"> Functional units of DNA.</p>
 
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<h3> Enhanced Part </h3>
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<h2>∆RuvCIII-2 ∆HNH ∆REC2 Sp-dCas9 Enhanced</h2>
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    <h3 class="w3-border-bottom w3-border-light-grey w3-padding-16">∆RuvCIII-2 ∆HNH ∆REC2 Sp-dCas9 Enhanced</h3>
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    <pstyle="text-align:center"> Validated Part </p>
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<p>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.</p>
 
<p>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.</p>
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<p>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 the various site of dCas9. Combination of quick change at Asn497Lys (QC5), Thr657Lys (QC6) and Ser1109Arg (QC15) show improved activity. Its binding capability was tested by assessing the strength in transcriptional activation via by tagging it with a VP64-p65-Rta tripartite activator.</p>
 
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Latest revision as of 02:55, 2 November 2017



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Basic Part

Functional units of DNA.

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

Validated Part

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.

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 the various site of dCas9. Combination of quick change at Asn497Lys (QC5), Thr657Lys (QC6) and Ser1109Arg (QC15) show improved activity. Its binding capability was tested by assessing the strength in transcriptional activation via by tagging it with a VP64-p65-Rta tripartite activator.