Microbiology

Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteria Leptothrix cholodnii SP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation with Escherichia coli S17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes.

Graphical overview

The unicellular red alga Cyanidioschyzon merolae has been used as a eukaryotic photosynthetic model for various basic and applied studies. Although the nuclear genome of C. merolae can be modified by homologous recombination with exogenously introduced DNA, it has been difficult to modify multiple chromosome loci within the same strain because of the limited number of available positive selection markers. Recently, we reported a modified URA5.3 gene cassette (URA5.3T), which can be used repeatedly for nuclear genome transformation using the pMKT plasmid vectors for epitope tagging (3x FLAG- or 3x Myc-) of nuclear-encoded proteins. In addition, these plasmid vectors can also be used to knock out multiple genes one by one. This report describes the construction of DNA fragments for transformation and the detailed transformation procedure.

The smut fungus Ustilago maydis is an established model organism for elucidating how biotrophic pathogens colonize plants and how gene families contribute to virulence. Here we describe a step by step protocol for the generation of CRISPR plasmids for single and multiplexed gene editing in U. maydis. Furthermore, we describe the necessary steps required for generating edited clonal populations, losing the Cas9 containing plasmid, and for selecting the desired clones.