ArticleMicrobial cell factories2008
Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes.
Article in Microbial cell factories, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 75 papers.
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Who cites it
75 citing papers in PubMed, 148 citations in OpenAlex.
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- A slight mismatch between a gene's codon usage and the cellular tRNA supply is beneficial.Nature communications · 2026Article
- Plasmid mutation rates scale with copy number.Proceedings of the National Academy of Sciences of the United States of America · 2026Article
- Beyond bactericidal: targeting plasmid-mediated antibiotic resistance with natural product-based plasmid curing agents.Frontiers in microbiology · 2026Review
- Construction and Characterization of Novel Shuttle Expression Vectors for Actinobacillus succinogenes.Biotechnology and applied biochemistry · 2025Article
- Deciphering plasmid replication dynamics: a computational approach to predict ColE1-like plasmid copy number.Journal of the Royal Society, Interface · 2025Article
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- Thermo-amplifier circuit in probiotic E. coli for stringently temperature-controlled release of a novel antibiotic.Journal of biological engineering · 2024Article
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- Plasmid-mediated phenotypic noise leads to transient antibiotic resistance in bacteria.Nature communications · 2024Article
- Minimizing endogenous cryptic plasmids to construct antibiotic-free expression systems forSynthetic and systems biotechnology · 2024Article
- Identification of the Major Facilitator Superfamily Efflux Pump KpsrMFS inInternational journal of molecular sciences · 2024Article
- Characterisation and mobilisation of IncA/C plasmid-mediated antibiotic resistance in Edwardsiella ictaluri.Journal of global antimicrobial resistance · 2023Article
- Characterization ofAntibiotics (Basel, Switzerland) · 2023Article
- Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125.Applied microbiology and biotechnology · 2023Article
- Revealing the Host-Dependent Nature of an Engineered Genetic Inverter in Concordance with Physiology.Biodesign research · 2023Article
- Inducible plasmid copy number control for synthetic biology in commonly used E. coli strains.Nature communications · 2022Article
- Deciphering the Regulatory Circuits of RA3 Replication Module - Mechanisms of the Copy Number Control.International journal of molecular sciences · 2022Article
15 more citing papers are in PubMed but not listed here.
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundRecombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed.
resultsTo avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95 degrees C for 10 minutes prior to storage at -20 degrees C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well) enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25 degrees C and the correlation between PCN and protein accumulation was established.
conclusionUsing whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.