title: Markerless Genome Editing in Competent Streptococci.
abstract: Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.
authors.affiliation: Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
authors.forename: Roger
authors.initials: R
authors.lastname: Junges
authors.affiliation: Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
authors.forename: Rabia
authors.initials: R
authors.lastname: Khan
authors.affiliation: Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois at Chicago, 845 West Taylor Street, (MC 066), Chicago, IL, 60607, USA.
authors.forename: Yanina
authors.initials: Y
authors.lastname: Tovpeko
authors.affiliation: Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
authors.forename: Heidi A
authors.initials: HA
authors.lastname: Åmdal
authors.affiliation: Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
authors.forename: Fernanda C
authors.initials: FC
authors.lastname: Petersen
authors.affiliation: Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois at Chicago, 845 West Taylor Street, (MC 066), Chicago, IL, 60607, USA. damorris@uic.edu.
authors.forename: Donald A
authors.initials: DA
authors.lastname: Morrison
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology; D000072669:Gene Editing; D005798:Genes, Bacterial; D005819:Genetic Markers; D016680:Genome, Bacterial; D009154:Mutation; D017384:Sequence Deletion; D012808:Sigma Factor / Q000378:metabolism / Q000494:pharmacology; D013291:Streptococcus / Q000235:genetics*; D014169:Transformation, Bacterial / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D005819:Genetic Markers; D012808:Sigma Factor; C423628:competence factor, Streptococcus
keywords: CSP; Competence; Markerless mutagenesis; Natural transformation; Pheromone; Streptococcus; Streptococcus mutans; Streptococcus pneumoniae; XIP