Difference between revisions of "Part:BBa K1645998:Design"

 
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===Design Notes===
 
===Design Notes===
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This part was cloned without the promoter to avoid the biobrick illegal site existing on the U6 promoter. You can refer Waterloo iGEM 2015 wiki to see the full sequence of the U6 promoter in order to clone it in. The characterization data using this part in our purposes suggests that our CRISPR cas9 system is working using this SgRNA by suppressing RFP which uses lacI promoter. Our experiment was done in BL21 and the SgRNA was in pSB3K3 backbone containing part BBa-K1645999. We were able to detect drop in RFP levels using this SgRNA coupled by S.pyrogenes dCas9.  
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This part was cloned without the promoter to avoid the biobrick illegal site existing on the U6 promoter. You can refer Waterloo iGEM 2015 wiki to see the full sequence of the U6 promoter in order to clone it in upstream of this part. The characterization data using this part in our purposes suggests that our CRISPR cas9 system is working using this sgRNA by suppressing RFP expression which uis under lacI promoter. Our experiment was done in BL21 and the sgRNA was in pSB3K3 backbone containing part BBa-K1645999. We were able to detect drop in RFP levels using this SgRNA coupled by S.pyrogenes dCas9.
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===Source===
 
===Source===

Latest revision as of 02:59, 26 September 2015


SgRNA targeting LacI promoter


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]


Design Notes

This part was cloned without the promoter to avoid the biobrick illegal site existing on the U6 promoter. You can refer Waterloo iGEM 2015 wiki to see the full sequence of the U6 promoter in order to clone it in upstream of this part. The characterization data using this part in our purposes suggests that our CRISPR cas9 system is working using this sgRNA by suppressing RFP expression which uis under lacI promoter. Our experiment was done in BL21 and the sgRNA was in pSB3K3 backbone containing part BBa-K1645999. We were able to detect drop in RFP levels using this SgRNA coupled by S.pyrogenes dCas9.

Source

The scaffold sequence was obtained from gRNA design protocol from Church Lab. Link is provided below: https://www.addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf

References