Tn5-rpsL: a new derivative of transposon Tn5 useful in plasmid curing

Stojiljković, Igor; Trgovčević, Željko; Salaj-Šmic, Erika

doi:10.1016/0378-1119(91)90039-e

Cited by 13 publications

(7 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An elegant solution is to use a transposon carrying a counterselectable marker. Tn5-derived mini-transposons, containing rpsL or sacB, were used to cure plasmids from Salmonella (51) and Rhizobium (18) strains in two-step selection protocols. First, a mutant in which the mini-transposon has integrated into the plasmid to be cured is selected either by DNA-DNA hybridization with the mini-transposon as a probe or by exconjugation.…”

Section: Selection Of Plasmid Alterationsmentioning

confidence: 99%

Counterselectable Markers: Untapped Tools for Bacterial Genetics and Pathogenesis

et al. 1998

View full text Add to dashboard Cite

Section: Selection Of Plasmid Alterationsmentioning

confidence: 99%

Counterselectable Markers: Untapped Tools for Bacterial Genetics and Pathogenesis

et al. 1998

View full text Add to dashboard Cite

“…Selectable markers are used for plasmid maintenance, engineered conjugation and genome manipulations (1). In contrast, counter-selectable markers such as sacB (2) or barnase (3) are useful tools for different applications, such as plasmid curing (4,5), scar-less gene deletion (2) or engineering double-crossovers (6). However, counter-selectable markers often require stringent growth conditions to achieve robust counter-selection performance, and there are few means to ensure proper function of counter-selectable markers in vivo , which limit their application.…”

Section: Introductionmentioning

confidence: 99%

Rational optimization of tolC as a powerful dual selectable marker for genome engineering

Gregg

Lajoie

Napolitano

et al. 2014

Nucleic Acids Research

View full text Add to dashboard Cite

Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices.

show abstract

“…One marker allowing the required negative selection is based on a common spontaneous bacterial streptomycin (Sm) resistance mutation in the gene rpsL that causes a lysine replacement in protein S12 of the small ribosomal subunit (21). As this mutation is recessive, an rpsL ϩ allele has been employed to provide a dominant drug-sensitive phenotype in genetic contexts where it can provide direct selection for deletion, mutation, or replacement events (5,7,8,17,20,(22)(23)(24)(25). Here we describe application of this principle for construction of an rpsL cassette for use in S. pneumoniae that allows use of antibiotics at both selection steps and show that it can be used with natural genetic transformation for gene replacement through negative selection.…”

mentioning

confidence: 99%

An rpsL Cassette, Janus, for Gene Replacement through Negative Selection in Streptococcus pneumoniae

Sung

Claverys

et al. 2001

Appl Environ Microbiol

375

405

View full text Add to dashboard Cite

Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL ؉ marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in S. pneumoniae. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sm resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.

show abstract

Tn5-rpsL: a new derivative of transposon Tn5 useful in plasmid curing

Cited by 13 publications

References 23 publications

Counterselectable Markers: Untapped Tools for Bacterial Genetics and Pathogenesis

Counterselectable Markers: Untapped Tools for Bacterial Genetics and Pathogenesis

Rational optimization of tolC as a powerful dual selectable marker for genome engineering

An rpsL Cassette, Janus, for Gene Replacement through Negative Selection in Streptococcus pneumoniae

Contact Info

Product

Resources

About