Laboratory Exercise to Measure Plasmid Copy Number by qPCR

Quantitative PCR (qPCR) has numerous applications in biology. In an education setting, qPCR provides students an opportunity to better understand the PCR mechanism by providing both quantitative information about the reactions and also data to troubleshoot PCRs (e.g., melt curves). Here, we present a relatively short (2-h) laboratory activity to demonstrate qPCR to quantify plasmid copy number (CN) by measuring the cycle threshold ( C T ) values for a genomic gene and a plasmid gene using transformed cells as a template. The activity can be combined with additional laboratory exercises, including bacterial transformation, to create the template to be used in the qPCRs. This lab activity is ideal for undergraduate laboratory courses that include recombinant DNA technology.

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Faculty Opinions recommendation of Increased plasmid copy number is essential for Yersinia T3SS function and virulence.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726475088.793562337 ◽

2019 ◽

Author(s):

Joan Mecsas

Keyword(s):

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy

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Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability

Molecular Biology and Evolution ◽

10.1093/molbev/msy225 ◽

2018 ◽

Vol 36 (3) ◽

pp. 472-486 ◽

Cited By ~ 18

Author(s):

Judith Ilhan ◽

Anne Kupczok ◽

Christian Woehle ◽

Tanita Wein ◽

Nils F Hülter ◽

...

Keyword(s):

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy

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Plasmid copy number noise in monoclonal populations of bacteria

Physical Review E ◽

10.1103/physreve.81.011909 ◽

2010 ◽

Vol 81 (1) ◽

Cited By ~ 18

Author(s):

Jérôme Wong Ng ◽

Didier Chatenay ◽

Jérôme Robert ◽

Michael Guy Poirier

Keyword(s):

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy

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Hypothesis: hypersensitive plasmid copy number control for ColE1

Biophysical Journal ◽

10.1016/s0006-3495(96)79555-3 ◽

1996 ◽

Vol 70 (1) ◽

pp. 135-145 ◽

Cited By ~ 9

Author(s):

M. Ehrenberg

Keyword(s):

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Copy Number Control

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Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02010-12 ◽

2013 ◽

Vol 57 (4) ◽

pp. 1850-1856 ◽

Cited By ~ 17

Author(s):

L. C. Cook ◽

G. M. Dunny

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Copy Number ◽

Antibiotic Resistance Genes ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Rapid Reduction ◽

Biofilm Growth ◽

Content Type

ABSTRACTBiofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity inEnterococcus faecaliscells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four differentE. faecalisplasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.

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