Coding

Part:BBa_K2876008

Designed by: Eleanor Glockner   Group: iGEM18_Stanford   (2018-10-10)


adCas9

aureus dCas9

Our project builds on a dual-dCas9 system by implementing a transcriptional-based reporter system by bridging dCas9 with the prokaryotic two-hybrid (P2H) system. In our system the proximity of dCas9 binding brings together a larger protein complex (adapted from the P2H system) and initiates transcription of red fluorescent protein (RFP).

We are adapting a prokaryotic two-hybrid system and combining it with dCas9 in order to selectively detect DNA sequences for disease diagnostics. In order to ensure specificity of sgRNA to dCas9 binding, we used two different dCas9 proteins derived from streptococcus pyogenes and streptococcus aureus bacteria. The pyogenes dCas9 was fused via a tri-alanine linker to a lambda repressor that binds the pOL-62 promoter region 35 base pairs upstream of the RFP initiation site. The aureus dCas9 was fused via a tri-alanine linker to the alpha-subunit of RNA polymerase, which in turn recruits polymerase for transcription. The two sgRNA sequences are produced separately and bind selectively to the dCas9 molecules and target DNA, thus bringing together the entire complex and activating RFP.

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 1193
    Illegal BglII site found at 1680
    Illegal BglII site found at 2303
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 139
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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