Construction and use of flow cytometry optimized plasmid-sensor strains.
Determining the stability of plasmids in bacterial populations is traditionally performed by isolating a large number of clones followed by screening for the presence of plasmids by replica transfer to plasmid-selective agar plates. This is often a laborious task, especially when the intrinsic stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid-harboring cells in a bacterial population facilitates a very high throughput of cells and thus provides excellent sensitivity and statistics toward detecting even very low levels of plasmid instability.
Bahl
Martin Iain
MI
Department of Microbiology, University of Copenhagen, Copenhagen, Denmark.
Oregaard
Gunnar
G
Sørensen
Søren J
SJ
Hansen
Lars Hestbjerg
LH
D001419:Bacteria / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D020563:Gammaproteobacteria / Q000235:genetics; D042822:Genomic Instability; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins