Plasmid Stability and L-Phenylalanine Production in Escherichia Coli

2018 ◽  
Vol 23 (4) ◽  
pp. 49-55
Author(s):  
Ha-Young Koh
Keyword(s):  
Escherichia Coli ◽  
Plasmid Stability

Fermentation strategy to enhance plasmid stability during the cultivation of Escherichia coli for the production of recombinant levansucrase

1998 ◽  
Vol 86 (4) ◽  
pp. 391-394 ◽  
Author(s):  
Chul Ho Kim ◽  
Jang Young Lee ◽  
Min Gon Kim ◽  
Ki Bang Song ◽  
Jeong Woo Seo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Stability ◽  
Fermentation Strategy

scholarly journals The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms

2020 ◽  
Vol 21 (2) ◽  
pp. 576 ◽  
Author(s):  
Luciana Gomes ◽  
Gabriel Monteiro ◽  
Filipe Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Plasmid Stability ◽  
Gene Dosage ◽  
Heterologous Protein Expression ◽  
Chemical Induction ◽  
T7 Promoter ◽  
Iptg Induction ◽  
The Impact

This work assesses the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of enhanced green fluorescent protein (eGFP) by planktonic and biofilm cells of Escherichia coli JM109(DE3) transformed with a plasmid containing a T7 promoter. It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Heterologous protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the heterologous protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of heterologous protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth.

scholarly journals Vertical and Horizontal Transmission of ESBL Plasmid from Escherichia coli O104:H4

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1207
Author(s):  
Sandra Daniel ◽  
Kelly Goldlust ◽  
Valentin Quebre ◽  
Minjia Shen ◽  
Christian Lesterlin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Stability ◽  
Horizontal Transmission ◽  
Viable Cell ◽  
Time Lapse ◽  
Conjugative Plasmid ◽  
Triple Mutant ◽  
Conjugational Transfer ◽  
Transposon Insertion ◽  
Free Cells

Multidrug resistance (MDR) often results from the acquisition of mobile genetic elements (MGEs) that encode MDR gene(s), such as conjugative plasmids. The spread of MDR plasmids is founded on their ability of horizontal transference, as well as their faithful inheritance in progeny cells. Here, we investigated the genetic factors involved in the prevalence of the IncI conjugative plasmid pESBL, which was isolated from the Escherichia coli O104:H4 outbreak strain in Germany in 2011. Using transposon-insertion sequencing, we identified the pESBL partitioning locus (par). Genetic, biochemical and microscopic approaches allowed pESBL to be characterized as a new member of the Type Ib partitioning system. Inactivation of par caused mis-segregation of pESBL followed by post-segregational killing (PSK), resulting in a great fitness disadvantage but apparent plasmid stability in the population of viable cells. We constructed a variety of pESBL derivatives with different combinations of mutations in par, conjugational transfer (oriT) and pnd toxin-antitoxin (TA) genes. Only the triple mutant exhibited plasmid-free cells in viable cell populations. Time-lapse tracking of plasmid dynamics in microfluidics indicated that inactivation of pnd improved the survival of plasmid-free cells and allowed oriT-dependent re-acquisition of the plasmid. Altogether, the three factors—active partitioning, toxin-antitoxin and conjugational transfer—are all involved in the prevalence of pESBL in the E. coli population.

scholarly journals Recovery of Plasmid pEMB1, Whose Toxin-Antitoxin System Stabilizes an Ampicillin Resistance-Conferring β-Lactamase Gene in Escherichia coli, from Natural Environments

2014 ◽  
Vol 81 (1) ◽  
pp. 40-47 ◽  
Author(s):  
Hyo Jung Lee ◽  
Hyun Mi Jin ◽  
Moon Su Park ◽  
Woojun Park ◽  
Eugene L. Madsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Stability ◽  
Functional Characterization ◽  
Treatment Plant ◽  
Antibiotic Resistance Genes ◽  
Cloning And Expression ◽  
Natural Environments ◽  
Ampicillin Resistance ◽  
Content Type ◽  
Lactamase Gene

ABSTRACTNon-culture-based procedures were used to investigate plasmids showing ampicillin resistance properties in two different environments: remote mountain soil (Mt. Jeombong) and sludge (Tancheon wastewater treatment plant). Total DNA extracted from the environmental samples was directly transformed intoEscherichia coliTOP10, and a single and three different plasmids were obtained from the mountain soil and sludge samples, respectively. Interestingly, the restriction fragment length polymorphism pattern of the plasmid from the mountain soil sample, designated pEMB1, was identical to the pattern of one of the three plasmids from the sludge sample. Complete DNA sequencing of plasmid pEMB1 (8,744 bp) showed the presence of six open reading frames, including a β-lactamase gene. Using BLASTX, theorf5andorf6genes were suggested to encode a CopG family transcriptional regulator and a plasmid stabilization system, respectively. Functional characterization of these genes using a knockoutorf5plasmid (pEMB1ΔparD) and the cloning and expression oforf6(pET21bparE) indicated that these genes were antitoxin (parD) and toxin (parE) genes. Plasmid stability tests using pEMB1 and pEMB1ΔparDEinE. colirevealed that theorf5andorf6genes enhanced plasmid maintenance in the absence of ampicillin. Using a PCR-based survey, pEMB1-like plasmids were additionally detected in samples from other human-impacted sites (sludge samples) and two other remote mountain soil samples, suggesting that plasmids harboring a β-lactamase gene with a ParD-ParE toxin-antitoxin system occurs broadly in the environment. This study extends knowledge about the dissemination and persistence of antibiotic resistance genes in naturally occurring microbial populations.

scholarly journals The Influence of Nutrient Medium Composition on Escherichia coli Biofilm Development and Heterologous Protein Expression

2021 ◽  
Vol 11 (18) ◽  
pp. 8667
Author(s):  
Alexandra Soares ◽  
Luciana C. Gomes ◽  
Gabriel A. Monteiro ◽  
Filipe J. Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Culture Media ◽  
Plasmid Stability ◽  
Epifluorescence Microscopy ◽  
Egfp Expression ◽  
Heterologous Protein Expression ◽  
E Coli ◽  
Biofilm Development

In the present study, the effects of different nutrient media on the development of Escherichia coli biofilms and the production of a heterologous protein were examined. E. coli JM109(DE3) cells transformed with pFM23 plasmid carrying the gene for enhanced green fluorescent protein (eGFP) expression were used. Cells were grown in two different culture media, Lysogenic Broth (LB) and M9ZB, in a flow cell system for 10 days. Epifluorescence microscopy, fluorimetry, and a high-performance liquid chromatography (HPLC) method based on hydrophobic interaction chromatography (HIC) were used to assess bacterial growth, plasmid copy number (PCN), and eGFP production in both planktonic and biofilm cells. The results showed that biofilm development was favored in M9ZB medium when compared with LB. However, the number of eGFP-expressing cells was higher in LB for both planktonic and sessile states (two-fold and seven-fold, respectively). In addition, the PCN in biofilm cells was slightly higher when using LB medium (on average, 29 plasmids per cell versus 20 plasmids per cell in M9ZB), and higher plasmid stability was observed in biofilms formed in LB compared to their planktonic counterparts. Hence, E. coli biofilms grown in LB enhanced both plasmid stability and capacity to produce the model heterologous protein when compared to M9ZB.

Plasmid Stability in PHA-Producing Recombinant Escherichia coli Strains

2006 ◽  
Vol 6 (5) ◽  
pp. 893-898
Author(s):  
Gustavo Graciano Fon . ◽  
Regina Vasconcellos .
Keyword(s):  
Escherichia Coli ◽  
Plasmid Stability ◽  
Recombinant Escherichia Coli
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