From ActinoBase
Revision as of 23:40, 24 March 2020 by H Otani (talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

A number of different methods have been developed for random and directed mutagenesis in actinobacteria (most notably for Streptomyces coelicolor). Many involve an initial mutagenesis step performed in Escherichia coli, with subsequent transfer of the mutant allele(s) into the desired strain i.e. by protoplast transformation or intergeneric conjugation into Streptomyces. Others, such as CRISPR/Cas- or meganuclease I-SceI- mediated genome editing, can be performed directly in the actinobacterial strain of interest.

Your choice of mutagenesis method will depend largely on your intended use, and on whether you have an appropriate genetic screen/selection that you can use to identify your mutant(s) of interest.

CRISPR/Cas9-mediated genome editing

Chemical mutagenesis

Depletion strains

Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")

Lambda-red mediated recombination using ssDNA

Meganuclease I-SceI based system for gene deletions

Protoplasts Formation, Regeneration and Transformation

"Quikchange" site-directed mutagenesis

Random mutagenesis using a mutator strain

Random mutagenesis using error-prone PCR

Suicide vectors


UV mutagenesis