PCRISPomyces-2: Difference between revisions
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*<em>[[Streptomyces lividans]]</em><sup>1</sup> | *<em>[[Streptomyces lividans]]</em><sup>1</sup> | ||
*<em>[[Streptomyces venezuelae]]</em> (unpulished mutagenesis performed within the group of [https://people.uea.ac.uk/m_hutchings Prof Matt Hutchings]) | *<em>[[Streptomyces venezuelae]]</em> (unpulished mutagenesis performed within the group of [https://people.uea.ac.uk/m_hutchings Prof Matt Hutchings]) | ||
*<em>[[Streptomyces viridochromogenes]]</em></em><sup>1</sup> | |||
Revision as of 13:33, 9 August 2019
Use
This plasmid can be used for precise CRISPR/Cas9-mediated genome engineering of Streptomyces strains1. It is possible to introduce mutations that range from 1-100kbp deletions, precise single base codon changes to alter amino acids and insertions to add Flag-tags to proteins encoded at their native loci.
This plasmid has been successfully used for genome editing in the following organisms
- Streptomyces albidoflavus2
- Streptomyces coelicolor (unpulished mutagenesis performed within the group of Prof Matt Hutchings)
- Streptomyces formicae3
- Streptomyces lividans1
- Streptomyces venezuelae (unpulished mutagenesis performed within the group of Prof Matt Hutchings)
- Streptomyces viridochromogenes1
See here for the original pCRISPomyces2 paper from Cobb et. al1.
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)
- Constitutive 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.
- Xba1 site for gibson assembly of repair templates
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
References
- Cobb R.E., Wang, Y., Zhao, H. (2014). High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synthetic Biology, 4(6), pp. 723-728. DOI: 10.1021/sb500351f
- McLean, T.C., Hoskisson, P.A., Seipke, R.F. (2016). Coordinate Regulation of Antimycin and Candicidin Biosynthesis. mSphere, 1(6), pp. e00305-e00316. DOI: 10.1128/mSphere.00305-16.
- Qin, Z., Munnoch, J.T., Devine R., Holmes, N.A., Seipke, R.F., Wilkinson, K.A., Wilkinson., Hutchings, M.H. (2017). Formicamycins, antibacterial polyketides produced by Streptomyces formicae isolated from African Tetraponera plant-ants. Chemical Science, 8, pp. 3218-3227. DOI: 10.1039/c6sc04265a