Accurate Determination of Plasmid Copy Number of Flow-Sorted Cells using Droplet Digital PCR

2014 ◽  
Vol 86 (12) ◽  
pp. 5969-5976 ◽  
Author(s):  
Michael Jahn ◽  
Carsten Vorpahl ◽  
Dominique Türkowsky ◽  
Martin Lindmeyer ◽  
Bruno Bühler ◽  
...  
Keyword(s):  
Copy Number ◽  
Accurate Determination ◽  
Digital Pcr ◽  
Plasmid Copy Number ◽  
Droplet Digital Pcr ◽  
Plasmid Copy

scholarly journals Spatiotemporal Variations in Growth Rate and Virulence Plasmid Copy Number during Yersinia pseudotuberculosis Infection

2020 ◽  
Author(s):  
Stephan Schneiders ◽  
Tifaine Hechard ◽  
Tomas Edgren ◽  
Kemal Avican ◽  
Maria Fällman ◽  
...  
Keyword(s):  
Growth Rates ◽  
Copy Number ◽  
Growth Dynamics ◽  
Digital Pcr ◽  
Plasmid Copy Number ◽  
Droplet Digital Pcr ◽  
Virulence Plasmid ◽  
Spatiotemporal Variations ◽  
Plasmid Copy ◽  
Infection Models

AbstractPathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Y. pseudotuberculosis up-regulates the virulence plasmid copy number (PCN) during infection and the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the current study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyer’s Patches and caecum during the clonal invasive phase of the infection, while the fastest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues, and will be readily applicable to other infection models.ImportanceStudying pathogenic bacteria proliferating inside infected hosts is challenging using traditional methods, especially the transit and reversible genetic events. The bacteria are effectively diluted by the overwhelming number of host cells present in infected tissues. Using an innovative droplet digital PCR (ddPCR) approach, we have determined the virulence plasmid copy number (PCN) variations and growth rates of Yersinia during the course of infection in a mouse model. Here, we show that both the virulence plasmid copy number and bacterial growth rates display spatiotemporal variations in mice during infection. We demonstrate that the peak-to-trough ratio can be used as a proxy for determining the growth rate of invasive bacterial pathogen during infection, and ddPCR as the method of choice for quantifying DNA in host-pathogen interaction context. This proof-of-concept ddPCR approach can be easily applied for any bacterial pathogens and any infection models, for analysis of PCN, growth dynamics and bacterial loads.

scholarly journals Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

PLoS ONE ◽  
2017 ◽  
Vol 12 (1) ◽  
pp. e0169846 ◽  
Author(s):  
Magdalena Plotka ◽  
Mateusz Wozniak ◽  
Tadeusz Kaczorowski
Keyword(s):  
Copy Number ◽  
Digital Pcr ◽  
Plasmid Copy Number ◽  
Droplet Digital Pcr ◽  
Plasmid Copy ◽  
Single Colour

scholarly journals Determination of Plasmid Copy Number Reveals the Total Plasmid DNA Amount Is Greater than the Chromosomal DNA Amount in Bacillus thuringiensis YBT-1520

PLoS ONE ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. e16025 ◽  
Author(s):  
Chunying Zhong ◽  
Donghai Peng ◽  
Weixing Ye ◽  
Lujun Chai ◽  
Junliang Qi ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Plasmid Dna ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Chromosomal Dna ◽  
Dna Amount ◽  
Plasmid Copy

Determination of plasmid copy number by fluorescence densitometry

Plasmid ◽  
1983 ◽  
Vol 9 (2) ◽  
pp. 182-190 ◽  
Author(s):  
Steven J. Projan ◽  
Stephen Carleton ◽  
Richard P. Novick
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy

scholarly journals Spatiotemporal Variations in Growth Rate and Virulence Plasmid Copy Number during Yersinia pseudotuberculosis Infection

2021 ◽  
Author(s):  
Stephan Schneiders ◽  
Tifaine Hechard ◽  
Tomas Edgren ◽  
Kemal Avican ◽  
Maria Fällman ◽  
...  
Keyword(s):  
Growth Rates ◽  
Copy Number ◽  
Yersinia Pseudotuberculosis ◽  
Gene Dosage ◽  
Growth Dynamics ◽  
Digital Pcr ◽  
Plasmid Copy Number ◽  
Virulence Plasmid ◽  
Mesenteric Lymph Nodes ◽  
Plasmid Copy

Pathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Y. pseudotuberculosis up-regulates the virulence plasmid copy number (PCN) during infection and the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the current study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyer’s Patches and caecum during the clonal invasive phase of the infection, while the fastest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues, and will be readily applicable to other infection models.

Rapid determination of plasmid copy number

1996 ◽  
Vol 49 (1-3) ◽  
pp. 219-229 ◽  
Author(s):  
Torsten Schmidt ◽  
Karl Friehs ◽  
Erwin Flaschel
Keyword(s):  
Copy Number ◽  
Rapid Determination ◽  
Plasmid Copy Number ◽  
Plasmid Copy

scholarly journals Determination of plasmid copy number and stability inClostridium acetobutylicumATCC 824

1993 ◽  
Vol 108 (3) ◽  
pp. 319-323 ◽  
Author(s):  
Sang Yup Lee ◽  
Lee D. Mermelstein ◽  
Eleftherios Terry Papoutsakis
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy
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