Balancing the Production of Two Recombinant Proteins inEscherichia coliby Manipulating Plasmid Copy Number: High-Level Expression of Heterodimeric Ras Farnesyltransferase

1997 ◽  
Vol 11 (3) ◽  
pp. 233-240 ◽  
Author(s):  
Kwei-Lan Tsao ◽  
David S Waugh
Keyword(s):  
Recombinant Proteins ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
High Level Expression ◽  
Plasmid Copy ◽  
Ras Farnesyltransferase ◽  
High Level

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 Alternative vanHAX promoters and increased vanA-plasmid copy number resurrect silenced glycopeptide resistance in Enterococcus faecium

2020 ◽  
Author(s):  
Theresa Maria Wagner ◽  
Jessin Janice ◽  
Audun Sivertsen ◽  
Ingegerd Sjögren ◽  
Arnfinn Sundsfjord ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Serial Transfer ◽  
Resistant Phenotype ◽  
High Level ◽  
Transplantation Patient ◽  
Copy Number Increase

Abstract Background Vancomycin variable enterococci (VVE) are van-positive isolates with a susceptible phenotype that can convert to a resistant phenotype during vancomycin selection. Objectives To describe a vancomycin-susceptible vanA-PCR positive ST203 VVE Enterococcus faecium isolate (VVESwe-S) from a liver transplantation patient in Sweden which reverted to resistant (VVESwe-R) during in vitro vancomycin exposure. Methods WGS analysis revealed the genetic differences between the isolates. Expression of the van-operon was investigated by qPCR. Fitness and stability of the revertant were investigated by growth measurements, competition and serial transfer. Results The VVESwe-R isolate gained high-level vancomycin (MIC >256 mg/L) and teicoplanin resistance (MIC = 8 mg/L). VVESwe-S has a 5′-truncated vanR activator sequence and the VVESwe-R has in addition acquired a 44 bp deletion upstream of vanHAX in a region containing alternative putative constitutive promoters. In VVESwe-R the vanHAX-operon is constitutively expressed at a level comparable to the non-induced prototype E. faecium BM4147 strain. The vanHAX operon of VVESwe is located on an Inc18-like plasmid, which has a 3–4-fold higher copy number in VVESwe-R compared with VVESwe-S. Resistance has a low fitness cost and the vancomycin MIC of VVESwe-R decreased during in vitro serial culture without selection. The reduction in MIC was associated with a decreased vanA-plasmid copy number. Conclusions Our data support a mechanism by which vancomycin-susceptible VVE strains may revert to a resistant phenotype through the use of an alternative, constitutive, vanR-activator-independent promoter and a vanA-plasmid copy number increase.

scholarly journals Plasmid Replicons for the Production of Pharmaceutical-Grade pDNA, Proteins and Antigens by Lactococcus lactis Cell Factories

2021 ◽  
Vol 22 (3) ◽  
pp. 1379
Author(s):  
Sofia O.D. Duarte ◽  
Gabriel A. Monteiro
Keyword(s):  
Lactococcus Lactis ◽  
Recombinant Proteins ◽  
Plasmid Copy Number ◽  
Fermented Food ◽  
Added Value ◽  
Regulatory Sequences ◽  
Plasmid Copy ◽  
Mucosal Vaccination ◽  
Cell Factories ◽  
Plasmid Replicons

The Lactococcus lactis bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination. In this review, we critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. A plasmid vector is an easily customized component when the goal is to engineer bacteria in order to produce a heterologous compound in industrially significant amounts, as an alternative to genomic DNA modifications. The additional burden to the cell depends on plasmid copy number and on the expression level, targeting location and type of protein expressed. For live mucosal vaccination applications, besides the presence of the necessary regulatory sequences, it is imperative that cells produce the antigen of interest in sufficient yields. The cell wall anchored antigens had shown more promising results in live mucosal vaccination studies, when compared with intracellular or secreted antigens. On the other side, engineering L. lactis to express membrane proteins, especially if they have a eukaryotic background, increases the overall cellular burden. The different alternative replicons for live mucosal vaccination, using L. lactis as the DNA vaccine carrier or the antigen producer, are critically reviewed, as a starting platform to choose or engineer the best vector for each application.

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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