scholarly journals Single-cell measurement of plasmid copy number and promoter activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bin Shao ◽  
Jayan Rammohan ◽  
Daniel A. Anderson ◽  
Nina Alperovich ◽  
David Ross ◽  
...  
Keyword(s):  
Protein Expression ◽  
Plasmid Dna ◽  
Copy Number ◽  
Promoter Activity ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Rna Transcripts ◽  
Cell Measurement ◽  
Single Living ◽  
Living Bacteria

AbstractAccurate measurements of promoter activities are crucial for predictably building genetic systems. Here we report a method to simultaneously count plasmid DNA, RNA transcripts, and protein expression in single living bacteria. From these data, the activity of a promoter in units of RNAP/s can be inferred. This work facilitates the reporting of promoters in absolute units, the variability in their activity across a population, and their quantitative toll on cellular resources, all of which provide critical insights for cellular engineering.

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

The effect of plasmid copy number mutations on pT181 replication initiator protein expression

Plasmid ◽  
1991 ◽  
Vol 25 (3) ◽  
pp. 198-207 ◽  
Author(s):  
Pei-Zhi Wang ◽  
Vitalia B. Henriquez ◽  
Steven J. Projan ◽  
Serban Iordanescu ◽  
Richard P. Novick
Keyword(s):  
Protein Expression ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Initiator Protein ◽  
Replication Initiator Protein ◽  
Replication Initiator

scholarly journals High-Level Production of Plasmid DNA by Escherichia coli DH5α ΩsacBby IntroducingincMutations

2014 ◽  
Vol 80 (23) ◽  
pp. 7154-7160 ◽  
Author(s):  
Ram Narayan Trivedi ◽  
Parvez Akhtar ◽  
Jonathan Meade ◽  
Patrick Bartlow ◽  
Mohammad M. Ataai ◽  
...  
Keyword(s):  
Mass Culture ◽  
Plasmid Dna ◽  
Copy Number ◽  
Minimal Medium ◽  
Antibiotic Resistance Gene ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Starting Point ◽  
High Level ◽  
Hydrolysis Of

ABSTRACTFor small-copy-number pUC-type plasmids, theinc1andinc2mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due toincmutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of theincmutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ∼1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C,inc2mutants of pNTC8485 exhibited a copy number of ∼7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use ofincmutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications.

scholarly journals Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability

2018 ◽  
Vol 36 (3) ◽  
pp. 472-486 ◽  
Author(s):  
Judith Ilhan ◽  
Anne Kupczok ◽  
Christian Woehle ◽  
Tanita Wein ◽  
Nils F Hülter ◽  
...  
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy

scholarly journals Plasmid copy number noise in monoclonal populations of bacteria

2010 ◽  
Vol 81 (1) ◽  
Author(s):  
Jérôme Wong Ng ◽  
Didier Chatenay ◽  
Jérôme Robert ◽  
Michael Guy Poirier
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy

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

2013 ◽  
Vol 57 (4) ◽  
pp. 1850-1856 ◽  
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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