Part:BBa_K2876009
alpha+adCas9
This composite part is an alpha subunit of RNApolIII fused to an 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 8
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1964
Illegal BglII site found at 2451
Illegal BglII site found at 3074 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 480
Illegal NgoMIV site found at 910 - 1000COMPATIBLE WITH RFC[1000]
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