PCRISPomyces-2: Difference between revisions
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*Codon optomised ''cas9'' gene under the constitutive ''rpSL'' promoter | *Codon optomised ''cas9'' gene under the constitutive ''rpSL'' promoter | ||
*LacZ cassette for golden gate cloning of guide RNA (gRNA) | *LacZ cassette for golden gate cloning of guide RNA (gRNA) | ||
*''gapdh | *''gapdh'' promoter for gRNA expression | ||
*''pSG5'' temparature sensitive ''Streptomyces'' origin of replication. Without selection and above 37°C the plasmid becomes unstable. This property is used to cure the plasmid from ''Streptomyces'' after successful mutagenesis. | *''pSG5'' temparature sensitive ''Streptomyces'' origin of replication. Without selection and above 37°C the plasmid becomes unstable. This property is used to cure the plasmid from ''Streptomyces'' after successful mutagenesis. | ||
Revision as of 10:28, 9 August 2019
Use
This plasmid can be used for precise CRISPR/Cas9-mediated genome engineering of Streptomyces strains. To our knowledge it has been successfully used in S. albidoflavus, S. coelicolor, S. formicae, S. lividans and S. venezuelae. Mutations have ranged from 1-100kbp deletions, precise codon changes to alter amino acids and insertions to add Flag-tags to proteins encoded at their native loci.
Click here for the CRISPR protocol used by Matt Hutchings lab (courtesy of Rebecca Devine) - or download the PDF.
Features
- Ampicillin resistance cassette
- Origin of conjugative transfer (oriT)
- Origin of replication for Escherichia coli (pBR322oriF)
- Codon optomised cas9 gene under the constitutive rpSL promoter
- LacZ cassette for golden gate cloning of guide RNA (gRNA)
- gapdh promoter for gRNA expression
- pSG5 temparature sensitive Streptomyces origin of replication. Without selection and above 37°C the plasmid becomes unstable. This property is used to cure the plasmid from Streptomyces after successful mutagenesis.
History
The plasmid was made by Huimin Zhao's group and is available free from AddGene under MTA: click here
Map
Sequence links
AddGene under MTA: click here
