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Differentially Marked IncP-1β R751 Plasmids for Cloning via Recombineering and Conjugation

ASHVEEN BAINS
ASHVEEN BAINS
 and 
JAMES W. WILSON
JAMES W. WILSON
   | Dec 05, 2019
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Article Category: short-communication
Published Online: Dec 05, 2019
Page range: 559 - 563
Received: Aug 20, 2019
Accepted: Oct 22, 2019
DOI: https://doi.org/10.33073/pjm-2019-052
Keywords
© 2019 Ashveen Bains and James W. Wilson et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Characterization of R751 plasmid derivatives.Panel A: Conjugation frequency (transconjugant per donor) of R751 derivatives compared to WT R751. Each conjugation was performed with different recipients with appropriate counterselective markers, and each R751 derivative is compared to the associated R751 control for that corresponding recipient performed simultaneously.
Characterization of R751 plasmid derivatives.Panel A: Conjugation frequency (transconjugant per donor) of R751 derivatives compared to WT R751. Each conjugation was performed with different recipients with appropriate counterselective markers, and each R751 derivative is compared to the associated R751 control for that corresponding recipient performed simultaneously.

Fig. 1.

Characterization of R751 plasmid derivatives.Panel B: Diagram of the FRT-Capture technique using plasmid R751 Sp as the cloning vehicle. The Sp-r marker can either be retained (via selection for Sp-r) or removed (by using solely Tp-r as the R751 plasmid selection) via this procedure (see text for details). Please, note that when the Sp-r marker is retained, insertion of the target DNA could occur on either side of the Sp-r marker (only one such insertion is shown). The insertion location can be easily verified using PCR or DNA sequencing of the plasmid.Panel C: Left-most picture: R751 Sp + pdu plasmid DNA was isolated and used as a template in PCR reactions using primers hybridizing to the pduW, pocR, and cobU genes. Primers hybridizing to the R751 Sp plasmid vector (kleE gene) were used as control. PCR products were run on 1.5% agarose and stained with SYBR Safe stain. The lanes labeled “1” and “2” are separate isolates of R751 Sp + pdu.Middle two pictures: E. coli TOP10 Rif strains containing either R751 Sp or R751 Sp + pdu were streaked onto MacConkey agar containing 1,2 PD as carbon source and supplemented with coenzyme B12. Red colony color indicates the expression of the pdu genes and metabolism of 1,2 PD. In addition, intact MCPs were isolated from TOP10 Rif (R751 Sp + pdu) and approximately 15 micrograms were run on an SDS-PAGE gel and stained with Coomassie. Asterisks on the gel photo indicate bands of known Pdu MCP proteins. Corresponding negative control strains display no bands (or a very faint non-MCP background band) via this analysis (data not shown).Right-most picture: R751 Cm + rimL plasmid DNA was isolated and used as a template in PCR reactions using primers hybridizing to the ydcO, rimL, and ydcS genes, and the samples were analyzed as above. The lanes labeled “1” and “2” are separate isolates of R751 Cm + rimL.
Characterization of R751 plasmid derivatives.Panel B: Diagram of the FRT-Capture technique using plasmid R751 Sp as the cloning vehicle. The Sp-r marker can either be retained (via selection for Sp-r) or removed (by using solely Tp-r as the R751 plasmid selection) via this procedure (see text for details). Please, note that when the Sp-r marker is retained, insertion of the target DNA could occur on either side of the Sp-r marker (only one such insertion is shown). The insertion location can be easily verified using PCR or DNA sequencing of the plasmid.Panel C: Left-most picture: R751 Sp + pdu plasmid DNA was isolated and used as a template in PCR reactions using primers hybridizing to the pduW, pocR, and cobU genes. Primers hybridizing to the R751 Sp plasmid vector (kleE gene) were used as control. PCR products were run on 1.5% agarose and stained with SYBR Safe stain. The lanes labeled “1” and “2” are separate isolates of R751 Sp + pdu.Middle two pictures: E. coli TOP10 Rif strains containing either R751 Sp or R751 Sp + pdu were streaked onto MacConkey agar containing 1,2 PD as carbon source and supplemented with coenzyme B12. Red colony color indicates the expression of the pdu genes and metabolism of 1,2 PD. In addition, intact MCPs were isolated from TOP10 Rif (R751 Sp + pdu) and approximately 15 micrograms were run on an SDS-PAGE gel and stained with Coomassie. Asterisks on the gel photo indicate bands of known Pdu MCP proteins. Corresponding negative control strains display no bands (or a very faint non-MCP background band) via this analysis (data not shown).Right-most picture: R751 Cm + rimL plasmid DNA was isolated and used as a template in PCR reactions using primers hybridizing to the ydcO, rimL, and ydcS genes, and the samples were analyzed as above. The lanes labeled “1” and “2” are separate isolates of R751 Cm + rimL.

Plasmids used in this study.

PlasmidReference
R751(Thorsted et al. 1998)
R751 Kmthis study
R751 Cmthis study
R751 Spthis study
pCP20(Datsenko and Wanner 2000)
pKD3(Datsenko and Wanner 2000)
pKD4(Datsenko and Wanner 2000)
pKD46(Datsenko and Wanner 2000)
pJW102(Quick et al. 2010)
eISSN:
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Language:
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Journal Subjects:
Life Sciences, Microbiology and Virology
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