Optimization of Proliferation Conditions of Recombinant Escherichia coli BL21(DE3)/pET-28b(+)-aroGM150

2014 ◽  
Vol 915-916 ◽  
pp. 887-890 ◽  
Author(s):  
Rong Liang ◽  
Chang Liu ◽  
Xiu Juan Meng ◽  
Song Yi Lin
Keyword(s):  
Escherichia Coli ◽  
Genetic Engineering ◽  
Recombinant Strain ◽  
Previous Experiment ◽  
Ph Value ◽  
Plasmid Stability ◽  
Hot Spot ◽  
Growth Characteristics ◽  
E Coli ◽  
Initial Ph

Applying Escherichia coli (E. coli) for fermentation is a very common technology. However combined with genetic engineering techniques to construct the recombinant Escherichia coli and study their growth characteristics has become the hot spot now. The recombinant Escherichia coli BL21 (DE3) /pET-28baroGM150 had been constructed by our laboratory in the previous experiment. And the purpose of this study was to optimize the proliferation conditions of Escherichia coli BL21 (DE3) /pET-28baroGM150. In order to make the recombinant Escherichia coli grow stable under suitable conditions, using the density of bacteria and plasmid stability as indexes, three factors were tested including temperature, initial pH and loading volume. And the results indicated that the optimal proliferation temperature of the recombinant strain was 30°C, initial pH value was 6.5, loading volume was 150 mL medium of 1000 mL bottles.

scholarly journals A One Health Comparative Assessment of Antimicrobial Resistance in Generic and Extended-Spectrum Cephalosporin-Resistant Escherichia coli from Beef Production, Sewage and Clinical Settings

2020 ◽  
Vol 8 (6) ◽  
pp. 885 ◽  
Author(s):  
Emelia H. Adator ◽  
Claudia Narvaez-Bravo ◽  
Rahat Zaheer ◽  
Shaun R. Cook ◽  
Lisa Tymensen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
One Health ◽  
Hot Spot ◽  
Municipal Sewage ◽  
Multi Drug Resistance ◽  
Beef Production ◽  
E Coli ◽  
Beef Processing ◽  
Extended Spectrum

This study aimed to compare antimicrobial resistance (AMR) in extended-spectrum cephalosporin-resistant and generic Escherichia coli from a One Health continuum of the beef production system in Alberta, Canada. A total of 705 extended-spectrum cephalosporin-resistant E. coli (ESCr) were obtained from: cattle feces (CFeces, n = 382), catch basins (CBasins, n = 137), surrounding streams (SStreams, n = 59), beef processing plants (BProcessing, n = 4), municipal sewage (MSewage; n = 98) and human clinical specimens (CHumans, n = 25). Generic isolates (663) included: CFeces (n = 142), CBasins (n = 185), SStreams (n = 81), BProcessing (n = 159) and MSewage (n = 96). All isolates were screened for antimicrobial susceptibility to 9 antimicrobials and two clavulanic acid combinations. In ESCr, oxytetracycline (87.7%), ampicillin (84.4%) and streptomycin (73.8%) resistance phenotypes were the most common, with source influencing AMR prevalence (p < 0.001). In generic E. coli, oxytetracycline (51.1%), streptomycin (22.6%), ampicillin (22.5%) and sulfisoxazole (14.3%) resistance were most common. Overall, 88.8% of ESCr, and 26.7% of generic isolates exhibited multi-drug resistance (MDR). MDR in ESCr was high from all sources: CFeces (97.1%), MSewage (96.9%), CHumans (96%), BProcessing (100%), CBasins (70.5%) and SStreams (61.4%). MDR in generic E. coli was lower with CFeces (45.1%), CBasins (34.6%), SStreams (23.5%), MSewage (13.6%) and BProcessing (10.7%). ESBL phenotypes were confirmed in 24.7% (n = 174) ESCr and 0.6% of generic E. coli. Prevalence of bla genes in ESCr were blaCTXM (30.1%), blaCTXM-1 (21.6%), blaTEM (20%), blaCTXM-9 (7.9%), blaOXA (3.0%), blaCTXM-2 (6.4%), blaSHV (1.4%) and AmpC β-lactamase blaCMY (81.3%). The lower AMR in ESCr from SStreams and BProcessing and higher AMR in CHumans and CFeces likely reflects antimicrobial use in these environments. Although MDR levels were higher in ESCr as compared to generic E. coli, AMR to the same antimicrobials ranked high in both ESCr and generic E. coli sub-populations. This suggests that both sub-populations reflect similar AMR trends and are equally useful for AMR surveillance. Considering that MDR ESCr MSewage isolates were obtained without enrichment, while those from CFeces were obtained with enrichment, MSewage may serve as a hot spot for MDR emergence and dissemination.

Study on Optimization of Fermentation Condition for Nitrilase-Producer Escherichia Coli BL21 (DE3)/pET-Nit

2011 ◽  
Vol 175-176 ◽  
pp. 192-196 ◽  
Author(s):  
Li Li Feng ◽  
Jian Fei Zhang ◽  
Hui Luo ◽  
Zheng Li ◽  
Hong Jie Zhang
Keyword(s):  
Escherichia Coli ◽  
Recombinant Strain ◽  
Culture Medium ◽  
Culture Conditions ◽  
Fermentation Condition ◽  
Hydrogen Phosphate ◽  
Strain Bl21 ◽  
Initial Ph ◽  
Potassium Hydrogen ◽  
Optimization Of Fermentation

The paper concentrated on the optimization of the recombinant strain BL21 (DE3)-PE7-Nit. The component of culture medium and the culture conditions were optimized. The optimized medium was: yeast extract 10 g/l, L-glutamate sodium 8 g/l, MgSO4.7H2O 0.7 g/l, Isopropyl-β-D-thiogalactopyranoside 0.3 mmol/L, potassium hydrogen phosphate 0.5 g / L, phosphate Potassium 0.5 g / L and the culture condition was: initial pH 7.0, inoculum 2%. The result showed that the activity of nitrilase prepared with these conditions increased by 130.37 % through optimization.

scholarly journals Inactivation of Escherichia coli and Staphyloccocus aureus in Litchi Juice by Dimethyl Dicarbonate (DMDC) Combined With Nisin

2014 ◽  
Vol 3 (3) ◽  
pp. 1 ◽  
Author(s):  
Yuanshan Yu ◽  
Yujuan Xu ◽  
Jijun Wu ◽  
Gengsheng Xiao ◽  
Jing Wen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Synergistic Effect ◽  
Ph Value ◽  
Inactivation Rate ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Complete Inactivation ◽  
Staphyloccocus Aureus ◽  
Addition Level

<p>Inactivation of Gram-negative <em>Escherichia coli</em> and Gram-positive <em>Staphyloccocus aureus</em> in litchi juice by DMDC combined with nisin was individually investigated. A 1.66 log cycles reduction of<em> E. coli </em>and 2.03 log cycles reduction of <em>S. aureus </em>in litchi juice (pH 4.5) added without nisin was achieved as exposed to 150 mg/l DMDC at 30 °C for 1 h, and the inactivation rate of <em>E. coli </em>and <em>S. aureus</em> during initial 1 h was far greater than during the remaining 5 h. As exposed to 150 mg/l DMDC at 30 °C for 1 h, the inactivation of <em>E. coli</em> and <em>S. aureus</em> in the litchi juice showed a trend toward increase with increasing of nisin addition level in the range from 0 to 200 IU/ml. Moreover, DMDC and nisin exhibited a synergistic effect on the inactivation of <em>E. coli</em> and <em>S. aureus</em> in litchi juice, and the inactivation of<em> E. coli</em> and <em>S. aureus</em> in the litchi juice also depends on the temperature of litchi juice, pH value of litchi juice and DMDC concentration when treated with DMDC and nisin. In addition, <em>E. coli</em> showed higher resistance to nisin as comparing with <em>S. aureus</em>. After <em>E. coli</em> and <em>S. aureus</em> in the litchi juice of pH 4.0 were individually treated with 150 mg/l DMDC combined with 200 IU/ml nisin at 30 °C for 1 h, a complete inactivation of <em>S. aureus</em> (6.59 log cycles) was achieved, but only 3.52 log cycles reduction of <em>E. coli</em> was observed.</p>

scholarly journals Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

2007 ◽  
Vol 74 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Alexander Mellmann ◽  
Shan Lu ◽  
Helge Karch ◽  
Jian-guo Xu ◽  
Dag Harmsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Integration Site ◽  
Hot Spot ◽  
Blot Hybridization ◽  
Enterohemorrhagic Escherichia Coli ◽  
Biologically Active ◽  
E Coli ◽  
Shiga Toxin 2

ABSTRACT Using colony blot hybridization with stx 2 and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx 2-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx 2-positive isolates fliC H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx 2-positive strains by loss of stx 2 during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx 2-positive and stx-negative isolates demonstrated that yecE was occupied in stx 2-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx 2-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx 2-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx 2-harboring phages. The ability to recycle stx 2, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.

scholarly journals Use of a Nonmedicated Dietary Supplement Correlates with Increased Prevalence of Streptomycin-Sulfa-Tetracycline-Resistant Escherichia coli on a Dairy Farm

2006 ◽  
Vol 72 (7) ◽  
pp. 4583-4588 ◽  
Author(s):  
Artashes R. Khachatryan ◽  
Thomas E. Besser ◽  
Dale D. Hancock ◽  
Douglas R. Call
Keyword(s):  
Escherichia Coli ◽  
Dietary Supplement ◽  
Dairy Farm ◽  
Washington State ◽  
Growth Characteristics ◽  
Control Group ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Dry Milk ◽  
Cell Densities

ABSTRACT We examined how a dietary supplement affects the prevalence of antibiotic-resistant Escherichia coli on a dairy farm in Washington State. Between 2001 and 2004 the prevalence of fecal E. coli strains resistant to streptomycin, sulfadiazine, and tetracycline (SSuT strains) declined from 59.2% to 26.1% in the calf population. In 2003 the dairy discontinued use of a dietary supplement, and we hypothesized that the decline in prevalence of SSuT strains was related to this change in management. To test this we established three treatments in which calves received no supplement, the dietary supplement with oxytetracycline, or the dietary supplement without oxytetracycline. Calves receiving either dietary supplement had a significantly higher prevalence of SSuT E. coli than the no-supplement control group (≈37% versus 20%, respectively; P = 0.03). Importantly, there was no evidence that oxytetracycline contributed to an increased prevalence of fecal SSuT E. coli. We compared the growth characteristics of SSuT and non-SSuT E. coli in LB broth enriched with either the complete dietary supplement or its individual constituents. Both the complete dietary supplement and its vitamin D component supported a significantly higher cell density of SSuT strains (P = 0.003 and P = 0.001, respectively). The dry milk and vitamin A components of the dietary supplement did not support different cell densities. These results were consistent with selection and maintenance of SSuT E. coli due to environmental components independent of antibiotic selection.

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.

Studies To Select Appropriate Nonpathogenic Surrogate Escherichia coli Strains for Potential Use in Place of Escherichia coli O157:H7 and Salmonella in Pilot Plant Studies†

2005 ◽  
Vol 68 (2) ◽  
pp. 282-291 ◽  
Author(s):  
DENISE R. EBLEN ◽  
BASSAM A. ANNOUS ◽  
GERALD M. SAPERS
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Time Lag ◽  
Growth Characteristics ◽  
Lag Phase ◽  
Phase Duration ◽  
Pathogenic Organism ◽  
Growth And Survival ◽  
E Coli ◽  
Genes Encoding

The response of a potential nonpathogenic surrogate organism to a particular treatment should closely mimic the response of the target pathogenic organism. In this study, growth characteristics (generation time, lag phase duration, and maximum population), pH at stationary phase, and survival characteristics (level of attachment and survival on apple surfaces, resistance to hydrogen peroxide decontamination treatments, and thermal resistance at 60°C) of 15 nonpathogenic generic Escherichia coli strains and one nonpathogenic E. coli O157:H43 strain were compared with those of two E. coli O157:H7 strains and two Salmonella strains. Few differences in growth characteristics or pH at stationary phase were evident between nonpathogenic and pathogenic strains tested. However, considerably more separation among strains was seen following investigation of survival characteristics. E. coli ECRC 97.0152, which does not contain genes encoding for known virulence factors associated with E. coli O157:H7, appears to be a good surrogate candidate, with growth and survival characteristics similar to those of E. coli O157:H7 strains. The less heat-resistant surrogate strains E. coli NRRL B-766 and NRRL B-3054 and E. coli ATCC 11775, ATCC 25253, and ATCC 25922 may be used when attempting to model the heat resistance of Salmonella Montevideo G4639 and Salmonella Poona RM 2350, respectively. These surrogate strains may be useful for evaluating the efficacy of intervention steps in reducing populations of selected strains of E. coli O157:H7 and Salmonella in processing environments where these pathogens cannot be introduced.

Fate of Enterohemorrhagic Escherichia coli O157: H7 in Commercial Mayonnaise

1994 ◽  
Vol 57 (9) ◽  
pp. 780-783 ◽  
Author(s):  
TONG ZHAO ◽  
MICHAEL P. DOYLE
Keyword(s):  
Escherichia Coli ◽  
Enterohemorrhagic Escherichia Coli ◽  
Health Concern ◽  
Plate Count ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Initial Ph ◽  
Contaminated Food ◽  
Aerobic Plate Count ◽  
Mold And Yeast

The fate of enterohemorrhagic Escherichia coli O157:H7 was determined in three different lots of commercial mayonnaise, including four different samples from a lot implicated in an outbreak of E. coli O157:H7 infection. The initial pH of the products ranged from 3.6 to 3.9. Products were inoculated with 6.5 × 103 E. coli O157:H7/g and incubated at 5 or 20°C. Escherichia coli O157:H7 did not grow at either temperature but survived for 34 to 55 days at 5°C and for 8 to 21 days at 20°C, depending on the lot. Survival was greatest in real mayonnaise purchased at retail among six mayonnaise samples which included a reduced calorie mayonnaise. Escherichia coli O157:H7 populations decreased between 2- and 100-fold by 3 weeks at 5°C, and between 10- and 1,000-fold by 7 days at 20°C. There was little or no change in pH (&lt;0.1 unit), aerobic plate count, mold and yeast count or Lactobacillus count (&lt; 1 log10 CFU/g) for the duration of the study. Commercial mayonnaise manufactured under good manufacturing practices is not a public health concern. Abusive handling of mayonnaise resulting in cross-contamination with E. coli O157:H7-contaminated food or contamination by an infected foodhandler is the principal basis for concern.

Killing of Escherichia coli using the gas diffusion electrode system

2010 ◽  
Vol 61 (1) ◽  
pp. 107-118
Author(s):  
W. Y. Xu ◽  
P. Li ◽  
B. Dong
Keyword(s):  
Escherichia Coli ◽  
Flow Rate ◽  
Current Density ◽  
Ph Value ◽  
Operating Cost ◽  
Gas Diffusion ◽  
Oxygen Flow Rate ◽  
Gas Diffusion Electrode ◽  
Oxygen Flow ◽  
E Coli

To be best of our knowledge, this study is one of the first investigations to be performed into the potential benefits of gas diffusion electrode (GDE) system in controlling inactivation of E. coli. This study mainly focused on the dual electrodes disinfection with gas diffusion cathode, using Escherichia coli as the indicator microorganisms. The effects of Pt load WPt and the pore-forming agent content WNH4HCO3 in GDE, operating conditions such as pH value, oxygen flow rate QO2, salt content and current density on the disinfection were investigated, respectively. The experimental results showed that the disinfection improved with increasing Pt load WPt, but its efficiency at Pt load of 3‰ was equivalent to that at Pt load of 4‰. Addition of the pore-forming agent in the appropriate amount improved the disinfection while drop of pH value resulted in the rapid rise of the germicidal efficacy and the disinfection shortened with increasing oxygen flow rate QO2. The system is more suitable for highly salt water. The germicidal efficacy increased with current density. However, the accelerating rate was different: it first increased with the current density, then decreased, and reached a maximum at current density of 6.7–8.3 mA/cm2. The germicidal efficacy in the cathode compartment was about the same as in the anode compartment indicating the contribution of direct oxidation and indirect treatment of E. coli by the hydroxyl radical was similar to the oxidative indirect effect of the generated H2O2. This technology is expensive in operating cost, further research is required to advance the understanding and reduce the operating cost of this technology.

scholarly journals Recent Advances in Shiga Toxin-Producing Escherichia coli Research in Latin America

2018 ◽  
Vol 6 (4) ◽  
pp. 100 ◽  
Author(s):  
Alfredo Torres ◽  
Maria Amaral ◽  
Leticia Bentancor ◽  
Lucia Galli ◽  
Jorge Goldstein ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Latin America ◽  
Latin American ◽  
Shiga Toxin ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Hot Spot ◽  
Research Progress ◽  
Health Issues ◽  
E Coli

Pathogenic Escherichia coli are known to be a common cause of diarrheal disease and a frequently occurring bacterial infection in children and adults in Latin America. Despite the effort to combat diarrheal infections, the south of the American continent remains a hot spot for infections and sequelae associated with the acquisition of one category of pathogenic E. coli, the Shiga toxin-producing E. coli (STEC). This review will focus on an overview of the prevalence of different STEC serotypes in human, animals and food products, focusing on recent reports from Latin America outlining the recent research progress achieved in this region to combat disease and endemicity in affected countries and to improve understanding on emerging serotypes and their virulence factors. Furthermore, this review will highlight the progress done in vaccine development and treatment and will also discuss the effort of the Latin American investigators to respond to the thread of STEC infections by establishing a multidisciplinary network of experts that are addressing STEC-associated animal, human and environmental health issues, while trying to reduce human disease. Regardless of the significant scientific contributions to understand and combat STEC infections worldwide, many significant challenges still exist and this review has focus in the Latin American efforts as an example of what can be accomplished when multiple groups have a common goal.

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