| Title: | Genome editing by natural genetic transformation in Streptococcus mutans |
| Author(s): | Morrison DA; Khan R; Junges R; Amdal HA; Petersen FC; |
| Address: | "Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois at Chicago, USA. Electronic address: DAMorris@uic.edu. Department of Oral Biology, Faculty of Dentistry, University of Oslo, Norway. Electronic address: rabia.khan@odont.uio.no. Department of Oral Biology, Faculty of Dentistry, University of Oslo, Norway. Electronic address: roger.junges@odont.uio.no. Department of Oral Biology, Faculty of Dentistry, University of Oslo, Norway. Electronic address: h.a.amdal@odont.uio.no. Department of Oral Biology, Faculty of Dentistry, University of Oslo, Norway. Electronic address: f.c.petersen@odont.uio.no" |
| DOI: | 10.1016/j.mimet.2015.09.023 |
| ISSN/ISBN: | 1872-8359 (Electronic) 0167-7012 (Linking) |
| Abstract: | "Classical mutagenesis strategies using selective markers linked to designed mutations are powerful and widely applicable tools for targeted mutagenesis via natural genetic transformation in bacteria and archaea. However, the markers that confer power are also potentially problematic as they can be cumbersome, risk phenotypic effects of the inserted genes, and accumulate as unwanted genes during successive mutagenesis cycles. Alternative mutagenesis strategies use temporary plasmid or cassette insertions and can in principle achieve equally flexible mutation designs, but design of suitable counter-selected markers can be complex. All these drawbacks are eased by use of direct genome editing. Here we describe a strategy for directly editing the genome of S. mutans, which is applied to the widely studied reference strain UA159 (ATCC 700610) and has the advantage of extreme simplicity, requiring construction of only one synthetic donor amplicon and a single transformation step, followed by a simple PCR screen among a few dozen clones to identify the desired mutant. The donor amplicon carries the mutant sequence and extensive flanking segments of homology, which ensure efficient and precise integration by the recombination machinery specific to competent cells. The recipients are highly competent cells, in a state achieved by treatment with a synthetic competence pheromone" |
| Keywords: | "*Genetic Techniques *Genome, Bacterial Mutation Recombination, Genetic Streptococcus mutans/*genetics *Transformation, Genetic Competence Markerless mutagenesis Natural transformation Pheromone;" |
| Notes: | "MedlineMorrison, D A Khan, R Junges, R Amdal, H A Petersen, F C eng Evaluation Study Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Netherlands 2015/10/21 J Microbiol Methods. 2015 Dec; 119:134-41. doi: 10.1016/j.mimet.2015.09.023. Epub 2015 Oct 19" |
