Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates

The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35 g l⁻¹ and 1.40 g l⁻¹ from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals.     

Quantitation of intracellular recombinant human superoxide dismutase using surface plasmon resonance

An immunosensor assay for the quantitation of intracellular recombinant human superoxide dismutase (rhSOD) in Escherichia coli cultivations based on detection with surface plasmon resoance (SPR) is described. A monoclonal antibody for rhSOD was immobilized on a SPR dextran gold chip. Bacterial samples were sonicated and centrifugated prior to injection over the antibody chip for SPR detection. The assay time was 7 min and allowed quantitation in the range of 1-64 nM SOD in lysate samples with a precision of 1.1-3.4%. The assay was applied to monitor the concentration of rhSOD during E. coli bioreactor cultivations where the rhSOD production was induced by iso-propyl-b-d-thiogalactoside (IPTG). The assay allowed accurate monitoring of the production of rhSOD where the important phases in the product formation were possible to see. The report also discusses influence from sample preparation, SPR selectivity and sensitivity and quantitation limits. The assay proved to be fast, sensitive and accurate with low background effects from the dextran matrix of the SPR chip.     

Dual gold nanoparticle lateflow immunoassay for sensitive detection of Escherichia coli O157:H7

Two patterns of signal amplification lateral flow immunoassay (LFIA), which used anti-mouse secondary antibody-linked gold nanoparticle (AuNP) for dual AuNP-LFIA were developed. Escherichia coli O157:H7 was selected as the model analyte. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP (anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP (anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose (NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-AuNP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 100-fold and reached 1.14 × 10³ CFU mL⁻¹. The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation.     

Molecular beacons: a real-time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. The development of a real-time polymerase chain reaction (PCR) assay to detect the presence of Escherichia coli using these fluorogenic reporter molecules is reported. MBs were designed to recognize a 19-bp region of the uid A gene, coding for an enzyme β-glucuronidase. The specificity of the MB-based PCR assay was evaluated for various E. coli strains as well as bacteria species that are present in nature. The capability of the assay to detect E. coli in drinking water and produce was demonstrated. Positive detection of E. coli was demonstrated when >10¹ CFU mL⁻¹ (colony forming unit) was present in the water samples and fresh produce after 18 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli in food and environmental samples.     

Combining biofunctional magnetic nanoparticles and ATP bioluminescence for rapid detection of Escherichia coli

A rapid, specific and sensitive method for assay of Escherichia coli (E. coli) using biofunctional magnetic nanoparticles (BMNPs) in combination with adenosine triphosphate (ATP) bioluminescence was proposed. The BMNPs were fabricated by immobilizing a specific anti-E. coli antibody on the surface of amine-functionalized magnetic nanoparticles (about 20 nm in diameter), and then was applied to capture the target bacteria E. coli from samples. The BMNPs exhibited high capture efficiency to E. coli. Transmission electron microscope (TEM) images showed that the BMNPs were bound to the surface of entire E. coli cells. The target bacteria became magnetic so that could be isolated easily from the sample solution by employing an external magnetic field. The concentration of E. coli captured by the BMNPs was then detected by an ATP bioluminescence method. The optimization of ATP measurement was carried out to improve the detection sensitivity. The proposed method was applied to detect the E. coli inoculated into pasteurized milk with low detection limit (20 cfu/mL) and short detection time (about 1 h).     

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H₂O₂) and H₂O₂-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H₂O₂ and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H₂O₂, as well as the incubation time between H₂O₂ and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10² CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10³ CFU/mL to 1.18 × 10⁶ CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control.     

Rapid determination of microorganisms using a flow-injection system

An automated method for the rapid determination of microorganisms using a flow-injection system is presented. Electrochemical measurement of a mediator reduced by microbial metabolism allowed the determination of fungi and bacteria in a few minutes. The lowest detection limit was 5 × 10⁶ colony-forming units (cfu) ml^?1 for Escherichia coli. Correlation between the flow-injection method and standard microbiological methods was excellent (r = 0.997, n = 4 for Beauveria bassiana; r = 0.997, n = 7 for E. coli). The flow-injection system was applied to the on-line control of an E. coli cultivation.     

Rapid detection of Escherichia coli O157:H7 spiked into food matrices

Food poisoning causes untold discomfort to many people each year. One of the primary culprits in food poisoning is Escherichia coli O157:H7. While most cases cause intestinal discomfort, up to 7% of the incidences lead to a severe complication called hemolytic uremic syndrome which may be fatal. The traditional method for detection of E. coli O157:H7 in cases of food poisoning is to culture the food matrices and/or human stool. Additional performance-based antibody methods are also being used. The NRL array biosensor was developed to detect multiple antigens in multiple samples with little sample pretreatment in under 30 min. An assay for the specific detection of E. coli O157:H7 was developed, optimized and tested with a variety of spiked food matrices in this study. With no sample pre-enrichment, 5 × 10³ cells mL⁻¹ were detected in buffer in less than 30 min. Slight losses of sensitivity (1-5 × 10⁻⁴ cell mL⁻¹⁾ but not specificity occur in the presence of high levels of extraneous bacteria and in various food matrices (ground beef, turkey sausage, carcass wash, and apple juice). No significant difference was observed in the detection of E. coli O157:H7 in typical culture media (Luria Broth and Tryptic Soy Broth).     

Multiplexed high-throughput electrokinetically-controlled immunoassay for the detection of specific bacterial antibodies in human serum

In previous studies we have developed a simple electrokinetically-controlled lab-on-a-chip for heterogeneous immunoassay. In that method, all the sequential operations in an immunoassay, such as reagent loading and washing, were performed automatically by electrokinetically controlling the flow in an H-shaped microchannel. Here, we demonstrated further development of a high-throughput immunoassay microfluidic chip, and the application of the new immunoassay microfluidic chip in assaying human serum. The microfluidic immunoassay analyzed ten samples in parallel in 22 min. Bacterial antibodies in samples were captured by antigens pre-patterned on the bottom wall of a microchannel and then bound with TRITC-labeled detection antibodies to generate fluorescent signals. With optimized surface concentration of antigen, the assay detected Escherichia coli O157:H7 antibody and Helicobacter pylori antibody from buffer solutions in concentration ranges of 0.02-10 μg mL⁻¹ and 0.1-50 μg mL⁻¹, respectively. Human sera that were E. coli-positive or H. pylori-positive were accurately distinguished from respective negative controls. Moreover, the two antibodies, anti-E. coli and anti- H. pylori antibodies, could be simultaneously detected from human serum. This electrokinetically-controlled immunoassay shows an excellent potential for efficiently detecting multiple pathogenic infections in clinical environments.     

High-Level Production of DNA-Specific Endonuclease AsEndI with Synonymous Codon and its Potential Utilization for Removing DNA Contamination

Endonuclease I is a widely distributed periplasmic or extracellular enzyme. A method for the high-level production of recombinant AsEndI (endonuclease I from Aliivibrio salmonicida) in Escherichia coli with secretion expression is investigated. The coding sequence of AsEndI gene was assembled according to the E. coli codon usage bias, and AsEndI was expressed in the periplasm of E. coli TOP10 with a C-terminal 6× His-tagged fusion. The recombinant AsEndI (His-AsEndI) was purified by Ni-NTA resin with a yield of 1.29?×?10⁷ U from 1-L LB medium. His-AsEndI could be classified into Ca²⁺/Mg²⁺-dependent nucleases and showed highest nuclease activity to dsDNA at pH 8.0 and 37 °C. His-AsEndI is highly active in a broad range of salt concentration range up to 1.0 M with optimal NaCl concentration at 0.4 M. His-AsEndI can effectively remove DNA contamination in RNA sample or in PCR reagents to the level that cannot be detected by highly sensitive nested PCR and without adverse effects on the subsequent PCR reaction. His-AsEndI can remove DNA contamination at high salt conditions, especially for the DNA that may be shielded by DNA-binding protein at low salt conditions.     

Fluorescence detection of Escherichia coli on mannose modified ZnTe quantum dots

Rapid detection and identification of Escherichia coli(E.coli) is essential to prevent its quickly spread.In this study,a novel fluorescence probe based on ZnTe quantum dots(QDs) modified by mannose(MAN)had been prepared for the determination of E.coli.The results showed that the obtained QDs showed excellent selectivity toward E.coli,and presented a good linearity in range of 1.0×10~5～1.0×10~8 CFU/mL.The optimum fluorescence intensity for detecting E. coli was found to be at pH 7.0 with a temperature of25℃ and incubation time of 20 min.Under these optimum conditions,the detection limit of E.coli was4.6×10~4 CFU/mL.The quenching was discussed to be a static quenching procedure,which was proved by the quenching efficiency of QDs decreased with the temperature increasing.     

Rapid detection of Escherichia coli by flow injection analysis coupled with amperometric method using an IrO2–Pd chemically modified electrode

An amperometric method for the rapid detection of Escherichia coli (E. coli) by flow injection analysis (FIA) using an IrO₂–Pd chemically modified electrode (CME) was developed in this paper. The method is based on a good marker β-d-galactosidase which was found in E. coli strains. β-d-galactosidase was produced by the induction of isopropyl β-d-thiogalactopyranoside (IPTG) and released from E. coli cells through the permeabilization of both polymyxin B nonapeptide and lysozyme to E. coli cells wall. The released β-d-galactosidase could catalyze the hydrolysis of the substrate p-aminophenyl β-d-galactopyranoside (PAPG) in the culture medium to produce 4-aminophenol which was proportional to the concentration of E. coli. Hence, E. coli could be detected by the determination of 4-aminophenol. An IrO₂–Pd CME, which showed high sensitivity in determination of 4-aminophenol, was prepared as the electro-detector in FIA. The amplified response current of 4-aminophenol obtained at the IrO₂–Pd CME was linear with the concentration of E. coli ranging from 2.0 × 10² to 1.0 × 10⁶ cfu/mL, the detection limit of this method to E. coli was 150 cfu/mL and the complete assay could be performed in 3 h.     

Chemluminescence immunoassay for estriol in saliva

Estriol is determined in whole saliva of pregnant women by a direct solid-phase chemiluminescence immunoassay. The assay uses a monclonal antibody raised against estriol-6-crboxymethyloxime/bovine serum albumin and the homologous chemiluminescent marker conjugate estriol- 6-carboxymethyloxime/aminopentylethylsiolominol (E3/APEI). The anti-estriol antibody is bound to the wells of a microtitration plate via a second antibody directed against the monoclonal antibody; 50 μl of saliva and 12.5 pg of E3/APEI per well are used. The incubation time is 10 min at room temperature. The calibration graph covers 5–750 pg of estriol and the detection limit is 4.5 pg (0.31 nmol l^-1. Mean recovery of added estriol is 98%. Within-assay coefficient of variation is 11.8–5% for 0.21–6.5 nmol l^-1 E3, and the between-assay value is 15.7–6.9% for 0.27-3.5 nmol l^-1 E3. The correlation of E3 concentration in time-matched samples of saliva and sera from pregnant women was good (r = 0.934). Total assay time including calculation of results is 3 h for 40 saliva samples.     

Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

The development and characterization of a magnetic bead (MB)-quantum dot (QD) nanoparticles based assay capable of quantifying pathogenic bacteria is presented here. The MB-QD assay operates by having a capturing probe DNA selectively linked to the signaling probe DNA via the target genomic DNA (gDNA) during DNA hybridization. The signaling probe DNA is labeled with fluorescent QD₅₆₅ which serves as a reporter. The capturing probe DNA is conjugated simultaneously to a MB and another QD₆₅₅, which serve as a carrier and an internal standard, respectively. Successfully captured target gDNA is separated using a magnetic field and is quantified via a spectrofluorometer. The use of QDs (i.e., QD₅₆₅/QD₆₅₅) as both a fluorescence label and an internal standard increased the sensitivity of the assay. The passivation effect and the molar ratio between QD and DNA were optimized. The MB-QD assay demonstrated a detection limit of 890 zeptomolar (i.e., 10⁻²¹ mol L⁻¹) concentration for the linear single stranded DNA (ssDNA). It also demonstrated a detection limit of 87 gene copies for double stranded DNA (dsDNA) eaeA gene extracted from pure Escherichia coli (E. coli) O157:H7 culture. Its corresponding dynamic range, sensitivity, and selectivity were also presented. Finally, the bacterial gDNA of E. coli O157:H7 was used to highlight the MB-QD assay's ability to detect below the minimum infective dose (i.e., 100 organisms) of E. coli O157:H7 in water environment.     

Surface plasmon resonance immunosensor for bacteria detection

This work describes an approach for the development of two bacteria biosensors based on surface plasmon resonance (SPR) technique. The first biosensor was based on functionalized gold substrate and the second one on immobilized gold nanoparticles. For the first biosensor, the gold substrate was functionalized with acid-thiol using the self-assembled monolayer technique, while the second one was functionalized with gold nanoparticles immobilized on modified gold substrate. A polyclonal anti-Escherichia coli antibody was immobilized for specific (E. coli) and non-specific (Lactobacillus) bacteria detection. Detection limit with a good reproducibility of 10⁴ and 10³ cfu mL⁻¹ of E. coli bacteria has been obtained for the first biosensor and for the second one respectively. A refractive index variation below 5 × 10⁻³ due to bacteria adsorption is able to be detected. The refractive index of the multilayer structure and of the E. coli bacteria layer was estimated with a modeling software.     

Piezoelectric Crystal Biosensor System for Detection of Escherichia Coli

Abstract

A piezoelectric crystal biosensor system was applied to the detection of Escherichia coli. the system consists of an oscillator, a frequency counter, a flow cell and a modified piezoelectric crystal. Anti-E. coli antibody is immobilized on the surface of the crystal. It is used as an E. coli detection by measuring its resonant frequency shift due to a mass change caused by specific binding of the micro organisms to the surface. the frequency shift correlates with an E. coli concentration in the range of 10⁶?10⁸ cells·cm^?3. the resonant frequency shift is increased by further treatment to bind micro-particles modified with anti-E. coli antibody. This method allows us to improve the determination limit to 10⁵ cells · cm^?3.     

Colorimetric detection of pathogenic bacteria using platinum-coated magnetic nanoparticle clusters and magnetophoretic chromatography

A colorimetric method that uses platinum-coated magnetic nanoparticle clusters (Pt/MNCs) and magnetophoretic chromatography is developed to detect pathogenic bacteria. Half-fragments of monoclonal Escherichia coli O157:H7 (EC) antibodies were functionalized to Pt/MNCs and used to capture E. coli bacteria in milk. After magnetic separation of free Pt/MNCs and Pt/MNC-EC complexes from the milk, a precision pipette was used to imbibe the E. coli-containing solution, then a viscous polyethylene glycol solution. Due to difference in viscosities, the solutions separate into two liquid layers inside the pipette tip. The Pt/MNC-EC complexes were separated from the free Pt/MNCs by applying an external magnetic field, then added to a tetramethylbenzidine (TMB) solution. Catalytic oxidation of TMB by Pt produced color changes of the solution, which enabled identification of the presence of 10 cfu mL⁻¹E. coli bacteria with the naked eye. The total assay time including separation, binding and detection was 30 min.     

Rapid identification of diarrheagenic Escherichia coli based on barcoded magnetic bead hybridization

Diarrhea, as a global public health problem, causes a large number of infections and deaths every year. Although Escherichia coli (E. coli) is one of the normal flora microorganisms in the human intestinal tract, it has five pathogenic bacteria types that can cause human diarrhea, known as diarrheagenic E. coli. When people are infected, rapid and accurate diagnosis, along with timely treatment, are especially important. Here, we introduce a new method to identify and analyze a large number of pathogenic strains in E. coli by multiplex PCR and barcoded magnetic bead hybridization. Results show that the detection sensitivities of enterohemorrhagic E. coli, enterotoxigenic E. coli, enteropathogenic E. coli, enteroinvasive E. coli and enteroaggregative E. coli were 1.3 × 10³ CFU/mL, 2 × 10⁴ CFU/mL, 4 × 10⁴ CFU/mL, 7.2 × 10⁴ CFU/mL and 1.7 CFU/mL respectively. This method has strong specificity and high sensitivity and detects multiple target sequences in one experiment. Compared with other methods, BMB array has great application potential.     

Antigenic specificity of Escherichia coli alkaline phosphatase studied with monoclonal antibodies: Immunological characterization of E. coli and Shigella strains

Monoclonal antibodies (MoAb) to the alkaline phosphatase of Escherichia coli were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of E. coli and SP₂O/Ag-14 myeloma cells. Five stable clones were established. They all produced antibodies which reacted by enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase of all E. coli (25 strains) independently of their origin (drinking water, saline water, surface water, faecal or clinical origin), and with that of four Shigella species (7 strains) tested. Four of these MoAb gave a positive reaction with 52 % (MoAb 4G10), 73 % (MoAb 4F8, MoAb 4G6) and 89 % (MoAb 3C8) of 14 other bacterial species (30 strains) studied, while one (MoAb 2E5) did not react with alkaline phosphatase of these unrelated bacterial strains and thus appeared specific for E. coli and Shigella species. This MoAb was still detectable in ascitic fluids at 1/500,000 in ELISA, and detected all E. coli strains in an indirect immunofluorescence assay at 1/100. It could therefore be used as a reagent for routine detection of E. coli in drinking water, food or clinical specimens.