scholarly journals A simple method for constructing magnetic Escherichia coli

2014 ◽  
Author(s):  
Yong-jun Lu ◽  
Mengyi Sun
Keyword(s):  
Escherichia Coli ◽  
Rational Design ◽  
Magnetic Force ◽  
Spatial Behavior ◽  
Simple Method ◽  
E Coli ◽  
Actual Application ◽  
Strain Bl21 ◽  
Different Shapes ◽  
Set Up

Magnetic force can serve as an ideal way to control the spatial behavior of microorganisms, because of its flexibility and penetrability. By incubation with the biocompatible compound, ammonium ferric citrate, as an iron source, we magnetized Escherichia coli, the most programmable chassis in synthetic biology. To enhance the magnetization efficiency, the ferritin protein, FtnA, from E. coli was cloned and overexpressed in strain BL21(DE3). The magnetization effect was observed within 30 min after harvest of bacteria, and the concentration of ammonium ferric acid used could be as low as 0.5 mM. Using different shapes of magnetic fields, different patterns could be generated easily. Our method may set up the foundation for a rational design of spatial structure of cell communities, which is important for their actual application.

Evaluation of biofilm performance as a protective barrier against biocorrosion using an enzyme electrode

2011 ◽  
Vol 64 (8) ◽  
pp. 1736-1742 ◽  
Author(s):  
S. Soleimani ◽  
B. Ormeci ◽  
O. B. Isgor ◽  
S. Papavinasam
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Microbiologically Influenced Corrosion ◽  
Simple Method ◽  
E Coli ◽  
Concrete Deterioration ◽  
Protective Barrier ◽  
Microbial Biofilms ◽  
Short Time ◽  
Selection Of

Sulfide is known to be an important factor in microbiologically influenced corrosion (MIC) of metals and concrete deterioration in wastewater treatment structures and sewer pipelines. A sulfide biosensor was used to determine the effectiveness of Escherichia coli DH5α biofilm as a protective barrier against MIC. The biofilm was shown to be effective in protecting surfaces from sulfide and helping to reduce MIC using amperometric measurements. The results also indicated that the growth conditions of E. coli DH5α may have an impact on the performance of the biofilm as a sulfide barrier. The simple method provided in this work enables the comparison of several microbial biofilms and selection of the ones with potential to prevent MIC in a relatively short time.

scholarly journals Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants of Escherichia coliCultures by Use of the Hemolysin System

2000 ◽  
Vol 66 (11) ◽  
pp. 5024-5029 ◽  
Author(s):  
Luis A. Fernández ◽  
Isabel Sola ◽  
Luis Enjuanes ◽  
Víctor de Lorenzo
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Culture Media ◽  
Single Chain ◽  
Extracellular Medium ◽  
Simple Method ◽  
E Coli ◽  
Single Chain Fv ◽  
Bacterial Cytoplasm ◽  
Culture Supernatants

ABSTRACT A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants ofEscherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.

scholarly journals In Silico Design for Systems-Based Metabolic Engineering In Escherichia Coli for The Bioconversion of Aerobic Glycerol to Succinic Acid

2020 ◽  
Author(s):  
Albert Enrique Tafur Rangel ◽  
Wendy Lorena Rios Guzman ◽  
Carmen Elvira Ojeda Cuella ◽  
Daissy Esther Mejia Perez ◽  
Ross Carlson ◽  
...  
Keyword(s):  
Machine Learning ◽  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Succinic Acid ◽  
In Silico ◽  
Rational Design ◽  
Industrial Biotechnology ◽  
E Coli ◽  
Cell Factories ◽  
Transcriptomics Data

Abstract BackgroundGlycerol has become an interesting carbon source for industrial processes as consequence of the biodiesel business growth since it has shown promising results in terms of biomass/substrate yields. Selecting the appropriate metabolic targets to build efficient cell factories and maximize the desired chemical production in as little time as possible is a major challenge in industrial biotechnology. The engineering of microbial metabolism following rational design has been widely studied. However, it is a cost-, time-, and laborious-intensive process because of the cell network complexity; thus, to be proficient is needed known in advance the effects of gene deletions.ResultsAn in silico experiment was performed to model and understand the effects of metabolic engineering over the metabolism by transcriptomics data integration. In this study, systems-based metabolic engineering to predict the metabolic engineering targets was used in order to increase the bioconversion of glycerol to succinic acid by Escherichia coli. Transcriptomics analysis suggest insights of how increase the glycerol utilization of the cell for further design efficient cell factories. Three models were used; an E. coli core model, a model obtained after the integration of transcriptomics data obtained from E. coli growing in an optimized culture media, and a third one obtained after integration of transcriptomics data obtained from E. coli after adaptive laboratory evolution experiments. A total of 2402 strains were obtained from these three models. Fumarase and pyruvate dehydrogenase were frequently predicted in all the models, suggesting that these reactions are essential to increasing succinic acid production from glycerol. Finally, using flux balance analysis results for all the mutants predicted, a machine learning method was developed to predict new mutants as well as to propose optimal metabolic engineering targets and mutants based on the measurement of importance of each knockout’s (feature’s) contribution.ConclusionsThe combination of transcriptome, systems metabolic modeling, and machine learning analyses revealed versatile molecular mechanisms involved in the utilization of glycerol. These data provide a platform to improve the prediction of metabolic engineering targets to design efficient cell factories. Our results may also work a guide platform for the selection/engineering of microorganisms for production of interesting chemical compounds.

Recirculating Immunomagnetic Separation and Optimal Enrichment Conditions for Enhanced Detection and Recovery of Low Levels of Escherichia coli O157:H7 from Fresh Leafy Produce and Surface Water

2007 ◽  
Vol 70 (12) ◽  
pp. 2717-2724 ◽  
Author(s):  
SUNEE HIMATHONGKHAM ◽  
MARY LEE DODD ◽  
JENNY K. YEE ◽  
DAVID K. LAU ◽  
RAYMOND G. BRYANT ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Surface Water ◽  
Real Time ◽  
Real Time Pcr ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Simple Method ◽  
E Coli ◽  
Low Levels ◽  
Spinach Leaves

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (CT values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42°C for 5 h provided better detection than enrichment at 37°C. Extended incubation of surface water for 20 h at 42°C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

scholarly journals Topical Application of Escherichia coli-Vectored Vaccine as a Simple Method for Eliciting Protective Immunity

2006 ◽  
Vol 74 (6) ◽  
pp. 3607-3617 ◽  
Author(s):  
Jianfeng Zhang ◽  
Zhongkai Shi ◽  
Fan-kun Kong ◽  
Edward Jex ◽  
Zhigang Huang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Topical Application ◽  
Defense Mechanism ◽  
Clostridium Tetani ◽  
Simple Method ◽  
Γδt Cells ◽  
E Coli ◽  
Innate Defense ◽  
Γδt Cell ◽  
Vectored Vaccine

ABSTRACT We report here that animals can be protected against lethal infection by Clostridium tetani cells and Bacillus anthracis spores following topical application of intact particles of live or γ-irradiated Escherichia coli vectors overproducing tetanus and anthrax antigens, respectively. Cutaneous γδT cells were rapidly recruited to the administration site. Live E. coli cells were not found in nonskin tissues after topical application, although fragments of E. coli DNA were disseminated transiently. Evidence suggested that intact E. coli particles in the outer layer of skin may be disrupted by a γδT-cell-mediated innate defense mechanism, followed by the presentation of E. coli ligand-adjuvanted intravector antigens to the immune system and rapid degradation of E. coli components. The nonreplicating E. coli vector overproducing an exogenous immunogen may foster the development of a new generation of vaccines that can be manufactured rapidly and administered noninvasively in a wide variety of disease settings.

scholarly journals Critical Synergistic Concentration of Lecithin Phospholipids Improves the Antimicrobial Activity of Eugenol against Escherichia coli

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Haoshu Zhang ◽  
Edward G. Dudley ◽  
Federico Harte
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Rational Design ◽  
Microbial Population ◽  
Critical Concentration ◽  
Antimicrobial Properties ◽  
Content Type ◽  
E Coli ◽  
Log Reduction ◽  
Naturally Occurring

ABSTRACT In this study, the effect of individual lecithin phospholipids on the antimicrobial properties of eugenol against Escherichia coli C600 was investigated. We tested five major phospholipids common in soy or egg lecithin (1,2-dihexadecanoyl-sn-glycero-3-phosphocholine [DPPC], 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine [DSPC], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-dihexadecanoyl-sn-glycero-3-phosphate [sodium salt] [DPPA], and 1,2-dihexadecanoyl-sn-glycero-3-phospho-l-serine [DPPS]) and one synthetic cationic phospholipid (1,2-dioctadecanoyl-sn-glycero-3-ethylphosphocholine [18:0 EPC]). Among the six phospholipids, DPPC, DSPC, DPPE, DPPA, and the cationic 18:0 EPC showed critical synergistic concentrations that significantly improved the inactivation effect of eugenol against E. coli after 30 min of exposure. At the critical synergistic concentration, an additional ca. 0.4 to 1.9 log reduction (ca. 0.66 to 2.17 log CFU/ml reduction) in the microbial population was observed compared to eugenol-only (control) treatments (ca. 0.25 log reduction). In all cases, increasing the phospholipid amount above the critical synergistic concentration (which was different for each phospholipid) resulted in antimicrobial properties similar to those seen with the eugenol-only (control) treatments. DPPS did not affect the antimicrobial properties of eugenol at the tested concentrations. The critical synergistic concentration of phospholipids was correlated with their critical micelle concentrations (CMC). IMPORTANCE Essential oils (EOs) are naturally occurring antimicrobials, with limited use in food due to their hydrophobicity and strong aroma. Lecithin is used as a natural emulsifier to stabilize EOs in aqueous systems. We previously demonstrated that, within a narrow critical-concentration window, lecithin can synergistically enhance the antimicrobial properties of eugenol. Since lecithin is a mixture of different phospholipids, we aimed to identify which phospholipids are crucial for the observed synergistic effect. This research studied the bioactivity of lecithin phospholipids, contributing to a rational design in using lecithin to effectively control foodborne pathogens in foods.

scholarly journals Simple Method for Plating Escherichia coli Bacteriophages Forming Very Small Plaques or No Plaques under Standard Conditions

2008 ◽  
Vol 74 (16) ◽  
pp. 5113-5120 ◽  
Author(s):  
Joanna M. Łoś ◽  
Piotr Golec ◽  
Grzegorz Węgrzyn ◽  
Alicja Węgrzyn ◽  
Marcin Łoś
Keyword(s):  
Escherichia Coli ◽  
Simple Method ◽  
E Coli ◽  
Low Concentrations

ABSTRACT The use of low concentrations (optimally 2.5 to 3.5 μg/ml, depending on top agar thickness) of ampicillin in the bottom agar of the plate allows for formation of highly visible plaques of bacteriophages which otherwise form extremely small plaques or no plaques on Escherichia coli lawns. Using this method, we were able to obtain plaques of newly isolated bacteriophages, propagated after induction of prophages present in six E. coli O157:H− strains which did not form plaques when standard plating procedures were employed.

scholarly journals Construction and evolution of anEscherichia colistrain relying on nonoxidative glycolysis for sugar catabolism

2018 ◽  
Vol 115 (14) ◽  
pp. 3538-3546 ◽  
Author(s):  
Paul P. Lin ◽  
Alec J. Jaeger ◽  
Tung-Yun Wu ◽  
Sharon C. Xu ◽  
Abraxa S. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Energy Metabolism ◽  
Rational Design ◽  
Minimal Medium ◽  
Global Regulation ◽  
Gene Deletions ◽  
Global Regulators ◽  
E Coli ◽  
Almost All ◽  
Tracer Experiments

The Embden–Meyerhoff–Parnas (EMP) pathway, commonly known as glycolysis, represents the fundamental biochemical infrastructure for sugar catabolism in almost all organisms, as it provides key components for biosynthesis, energy metabolism, and global regulation. EMP-based metabolism synthesizes three-carbon (C3) metabolites before two-carbon (C2) metabolites and must emit one CO2in the synthesis of the C2 building block, acetyl-CoA, a precursor for many industrially important products. Using rational design, genome editing, and evolution, here we replaced the native glycolytic pathways inEscherichia coliwith the previously designed nonoxidative glycolysis (NOG), which bypasses initial C3 formation and directly generates stoichiometric amounts of C2 metabolites. The resulting strain, which contains 11 gene overexpressions, 10 gene deletions by design, and more than 50 genomic mutations (including 3 global regulators) through evolution, grows aerobically in glucose minimal medium but can ferment anaerobically to products with nearly complete carbon conservation. We confirmed that the strain metabolizes glucose through NOG by13C tracer experiments. This redesignedE. colistrain represents a different approach for carbon catabolism and may serve as a useful platform for bioproduction.

scholarly journals Decreasing the Level of Ethyl Acetate in Ethanolic Fermentation Broths of Escherichia coli KO11 by Expression of Pseudomonas putida estZ Esterase

2002 ◽  
Vol 68 (6) ◽  
pp. 2651-2659 ◽  
Author(s):  
Adnan Hasona ◽  
S. W. York ◽  
L. P. Yomano ◽  
L. O. Ingram ◽  
K. T. Shanmugam
Keyword(s):  
Escherichia Coli ◽  
Ethyl Acetate ◽  
Pseudomonas Putida ◽  
Esterase Activity ◽  
Fermentation Broth ◽  
Serine Esterase ◽  
Ph Change ◽  
Simple Method ◽  
E Coli ◽  
Naphthyl Acetate

ABSTRACT During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter−1. Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using α-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40°C. The Km and V max for α-naphthyl acetate were 18 μM and 48.1 μmol · min−1 · mg of protein−1, respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 μmol · min−1 · mg of protein−1), followed by ethyl acetate (66 μmol · min−1 · mg of protein−1). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter−1.

scholarly journals Making All Parts of the 16S rRNA of Escherichia coli Accessible In Situ to Single DNA Oligonucleotides

2006 ◽  
Vol 72 (1) ◽  
pp. 733-744 ◽  
Author(s):  
L. Şafak Yilmaz ◽  
Hatice E. Ökten ◽  
Daniel R. Noguera
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Fluorescence Intensity ◽  
Rational Design ◽  
Mechanistic Model ◽  
Mechanistic Modeling ◽  
E Coli ◽  
Dna Oligonucleotides ◽  
Nucleic Acid Interactions

ABSTRACT rRNA accessibility is a major sensitivity issue limiting the design of working probes for fluorescence in situ hybridization (FISH). Previous studies empirically highlighted the accessibility of target sites on rRNA maps by grouping probes into six classes according to their brightness levels. In this study, a recently proposed mechanistic model of FISH, based on the thermodynamics of secondary nucleic acid interactions, was used to evaluate the accessibility of the 16S rRNA of Escherichia coli to fluorescein-labeled oligonucleotides when thermodynamic and kinetic barriers were eliminated. To cover the entire 16S rRNA, 109 probes were designed with an average thermodynamic affinity (ΔG o overall) of −13.5 kcal/mol. Fluorescence intensity was measured by flow cytometry, and a brightness threshold between classes 3 and 4 was used as the requirement for proof of accessibility. While 46% of the probes were above this threshold with conventional 3-h hybridizations, extending the incubation period to 96 h dramatically increased the fraction of bright probes to 86%. Insufficient thermodynamic affinity and/or fluorophore quenching was demonstrated to cause the low fluorescence intensity of the remaining 14% of the probes. In the end, it was proven that every nucleotide in the 16S rRNA of E. coli could be targeted with a bright probe and, therefore, that there were no truly inaccessible target regions in the 16S rRNA. Based on our findings and mechanistic modeling, a rational design strategy involving ΔG o overall, hybridization kinetics, and fluorophore quenching is recommended for the development of bright probes.

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