Part:BBa_K2644009
dcas9+ω
A gene for dcas9 protain along with ωbinding region, which can improve the level of transcription.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1099
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3378
Illegal BamHI site found at 4212 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage
A sequence for expressing dcas-ω protein. Dcas9 protein is a Cas9 catalytic site mutant, and it loses the ability to cut DNA strands . Dcas9-ωis constructed by fusing dcas9 to the omega subunit of RNAP. By dcas-ω directing to promoter regions of target genes, this strategy can enhance transcription. And we get the sequence through buying the plasmid pwj66 on the addgene website. In our project, dcas-ω is directed to asrR promoter to enhance the expression of report protein smURFP to improve sensitivity of arsenic ions detection.
Biology
Dcas9-ωis constructed by fusing dcas9 to the omega subunit of RNAP. It can be used to activate gene expression, with the possibility of achieving different levels of activation depending on the strength of the targeted promoter. To achieve its function, we need design appropriate spacers and replace the crRNA sequnce of pwj66 plasmid with them. The spacers should be in the upstream of the targeted promoter and the effect relates to the distance between PAM and the promoter. After dcas-ω is expressed in vivo, sgRNA will direct it to the corresponding place of the gene to enhance expression.
Reference
[1]David Bikard, Wenyan Jiang, Poulami Samai, Ann Hochschild , Feng Zhang, Luciano A. Marraffini. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. [J].Nucleic Acids Research, 2013, Vol. 41, No. 15 7429–7437
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