Protocols

Protocols organised by category

Bacteriophage

• Streptomyces Bacteriophage plaque assay

• High titre preparation of phage

• Isolation of phages from single plaques

• Isolation of phages from environmental samples

Biochemistry

• GUS assay (β-Glucuronidase assay)

Microbiology

• General Characterisaton of Actinomycetes

• Conjugation using ET12567/pUZ8002

• Streptomyces Exploration

• General Growth and Maintenance of Streptomyces sp.

• Isolating Actinomycetes from the environment

For a comprehensive guide to actinomycete characterisation see the Compendium of Actinobacteria from Prof. Joachim Wink at the German Collection of Microorganisms & Cell Cultures (DSMZ). This resouce provides protocols for characterising a wide range of features for new isolates, but may also be applicable more broadly for example for phenotyping experiments. The tests include colony morphology, melanin production, salt tolerance, carbon utlilisation and much more. To download the guide please see the link below.

DSMZ Compendium of Actinobacteria

Plant-Microbe interactions

• See main section for full list of protocols

Microscopy

• Staining of Streptomyces spp. Cell Wall and Nucleic Acids

• Cultivating Actinobacteria for Microscopic Analysis

Mutagenesis

• CRISPR/Cas9-mediated genome editing

• Chemical mutagenesis

• CUBIC

• 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

• Transposons

• UV mutagenesis

Natural Products

• Cloning, Transformation and Expression of Gene Clusters

• Metabolite extractions

• Production of microbial volatile organic compounds

Next Generation Sequencing

• ChIP Sequencing
• ChIP-exo sequencing

https://www.nature.com/articles/s41467-018-05265-7/

• RNA Sequencing
• rRNA depletion with Streptomyces coelicolor A3(2) specific probes
• Differential RNA sequencing

Nucleic Acid Methods

• Codon Optimising Sequences for Heterologous Expression in Streptomyces

• High GC PCR

• RNA extraction from Actinobacteria

• RNA interference

• Salting Out Genomic DNA Extraction Method

• Southern Blotting

Protein Methods

• Isolating extracellular protein from Streptomyces

• Protein production in Streptomyces

Alphabetical list of protocols

Characterisation of Actinomycetes

Cloning, Transformation and Expression of Gene Clusters

CRISPR/Cas9-mediated genome editing

Chemical mutagenesis

ChIP Seq

Codon Optimising Sequences for Heterologous Expression in Streptomyces

Conjugation using ET12567/pUZ8002

Depletion strains

Streptomyces Exploration

General Growth and Maintenance of Streptomyces sp.

GUS assay (β-Glucuronidase assay)

High GC PCR

High titre preparation of phage

Isolating Actinomycetes from the environment

Isolating extracellular protein from Streptomyces

Isolating Streptomyces from plant roots

Isolation of phages from environmental samples

Isolation of phages from single plaques

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

Metabolite extractions

Protoplasts Formation, Regeneration and Transformation

"Quikchange" site-directed mutagenesis

Random mutagenesis using a mutator strain

Random mutagenesis using error-prone PCR

RNA extraction from Actinobacteria

Salting Out Genomic DNA Extraction Method

Southern Blotting

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

Streptomyces Bacteriophage plaque assay

Suicide vectors

Transposons

UV mutagenesis