University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Website: @unf

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Classification: Bacteria -> escherichia coli

Citations 14

"Detection Of Bacterial ATP By Reversed Flow Injection Analysis With Luminescence Detection"
Anal. Chim. Acta 1992 Volume 266, Issue 2 Pages 339-343
J. N. Miller* and M. B. Nawawi, C. Burgess

Abstract: Cells of Escherichia coli were treated with lysozyme or an ultrasonic cell disintegrator and the resulting ATP solution was mixed in a flow injection manifold (diagram given) with a solution of luciferase, D-luciferin, bovine serum albumin and 0.5 M Mg2+ in a carrier stream of 0.02 M Tris - HCl (pH 7.75). A liquid scintillation counter was used for detection. The calibration graph was approximately rectilinear up to 1 nM-ATP with limits of detection of ~0.01 to 0.1 pM. Some common pharmaceuticals (nitrazepam, phenacetin, ephedrine) interfered only at relatively high concentration. Flow injection analysis with luminometric detection was used to detect trace concentrations. of ATP released from bacteria. Luciferin and luciferase reagents were injected into a flowing stream of aqueous ATP or of bacterial ext. A liquid scintillation counter was used as the detector. Experimental variables were studied, and 120 samples/h could be analyzed. In pure solution, ATP was detected at the 10^-14 M level. Because of incomplete extraction of the cellular ATP and the quenching effects of co-extracted materials, the limit of detection for the bacterial extracts was much poorer at about 10^3 - 10^4 cells/mL, corresponding to about 10-^11 - 10-^10 M ATP.
ATP Luminescence Scintillation counter Interferences Reverse

"Bypass Trapped Flow Analysis System (ByT-FAS) Used In Application: Quantitative Chemiluminescent Detection Of Whole Intact E. Coli Cell Genetic Transcription Levels Via Induction Of Luciferase With Tetracycline"
Talanta 1998 Volume 45, Issue 3 Pages 513-518
Stephen W. Hillard* and Kent K. Stewart

Abstract: Bypass trapped flow anal. system (ByT-FAS) is a new trapped flow app. designed to perform chemical measurements with flow injection systems at phys. steady state using small volumes (10-150 µL) of injected sample and reagent. A micro-volume version of ByT-FAS instrumentation with a chemiluminescent detection system was used to quantitate the protein transcription levels of transformed whole intact E. coli cells. The cells were transformed using a firefly luciferase encoding plasmid with a tetracycline inducible promoter. Luciferase synthesis was induced in E. coli cells containing multiple copies of this plasmid by different levels of tetracycline in the growth media. The level of induction was determined by measuring the velocity of luciferase enzyme per absorbance unit of the injected culture. The micro-volume ByT-FAS instrumentation permitted the rapid determination of the level of induced luciferase and was significantly faster than the traditional quant. determination of genetic transcription levels. The micro-volume ByT-FAS assays also used significantly lower amounts of the expensive substrate luciferin. This is the first reported use of ByT-FAS for the detection and analyzes of transformed cells. ByT-FAS with chemiluminescent detection has the potential of being a useful tool for the rapid analyzes of promoter DNA sequences, promoters, and transcription repressors in whole intact bacterial cells by mol. biologists and biochemists.
Chemiluminescence Bypass trapped

"Assay Of Biological Thiols By A Combination Of High Performance Liquid Chromatography And Post-column Reaction With 6,6'-dithiodi(nicotinic Acid)"
Anal. Biochem. 1984 Volume 138, Issue 1 Pages 95-98
Junko Nishiyama and Toyo Kuninori

Abstract: Thiols were separated by HPLC on a reversed-phase column (25 cm x 4.6 mm) packed with Fine Sil C18-10, with, as mobile phase, 33 mM KH2PO4 adjusted to pH 2.2 with H3PO4 or 33 mM sodium phosphate of pH 6.8; detection was by post-column derivatization with 6,6'-dithiodi(nicotinic acid) and measurement of the absorbance of the released 6-mercaptonicotinic acid at 344 nm. A comparison was made with post-column derivatization with 5,5'-dithiobis-(2-nitrobenzoic acid). Cysteine, cysteamine, homocysteine, glutathione and penicillamine were determined (detection limit 0.1 nmol); ergothioneine, 2-thiouracil and thiolhistidine could be separated but not determined. The method was applied to the assay of glutathione in human erythrocytes and in E. coli.
Cysteine Cysteamine Homocysteine Glutathione Penicillamine HPLC Spectrophotometry Post-column derivatization

"Determination Of Protein Expression And Plasmid Copy Number From Cloned Genes In Escherichia Coli By Flow Injection Analysis Using An Enzyme Indicator Vector"
Biotechnol. Bioeng. 1989 Volume 34, Issue 8 Pages 1023-1036
F. J. Schendel, E. J. Baude, M. C. Flickinger

Abstract: On-line determination of expression rates from cloned genes in Escherichia coli and of plasmid copy number would be useful for monitoring accumulation of non-secreted proteins. As an initial model for monitoring gene expression in intact cells, a non-gene-fusion enzyme-based indicator plasmid has been constructed containing the phoA gene coding for alkaline phosphatase (AP) in pUCIS and pACYC184. The activity of AP can be rapidly determined in permeabilized cells. A flow injection analysis (FIA) assay has been developed which allows the direct real-time measurement of the AP activity during cell growth. A model target gene coding for E. coli cyanase (cynS) has been inserted in order to determine the ratio between the expression of the target and indicator, AP. A linear relationship has been found between plasmid copy number and AP activity for the high-copy pUC vector. To minimize indicator expression, transcription terminators have been inserted between the cynS and phoA genes, altering the target-to-indicator ratio by 10- to 40-fold. These vectors may be useful for the rapid continuous determination of plasmid copy number and target gene expression for nonsecreted proteins and would overcome the limitations of in situ probe biosensors for real-time determination of the accumulation of proteins from cloned genes in E. coli.
Protein Enzyme

"Simultaneous Online Monitoring Of Intracellular β-galactosidase Activity And Biomass Using Flow Injection Analysis In Escherichia Coli Batch Fermentations"
Biotechnol. Techniq. 1992 Volume 6, Issue 3 Pages 213-218
Valero F., Lafuente F. J., Solà C., Benito A., Vidal M., Cairó J. and Villaverde A.

Abstract: Fermentation sample was sonicated at 4°C for 45 s and subjected to flow injection analysis after mixing with phosphate buffer solution (pH 7.0) containing 2-β-mercaptoethanol. The solution was mixed with 2-nitrophenyl-β-D-galactopyranoside at 47°C before addition of 1 M Na2CO3 and 0.5% Na dodecyl sulfate and detection at 420 nm. Biomass determination was achieved offline at 550 nm. The limit of detection was 25 iu mL-1 of β-galactosidase and the calibration graph was rectilinear for 5100 iu mL-1. The coefficient of variation was 1.5%. A novel method to monitor online intracellular β-galactosidase activity and biomass simultaneously, using flow injection analysis (FIA), was developed. The automatic ultrasonic cell disruption and FIA anal. allow the processing of 10 samples/h with a wide and variable linear working range of β-galactosidase activity and biomass and a max. relative standard deviation of 1.5%. The system was optimized by monitoring biomass and intracellular β-galactosidase activity in E. coli batch fermentation
Enzyme, galactosidase Spectrophotometry Process monitoring

"Measurement Of Intracellular Adenosine Triphosphate Using An Online FIA System For Determination Of Micro-organism Cells Number"
Bunseki Kagaku 1989 Volume 38, Issue 11 Pages T183-T186
Haketa, Y.;Motohashi, R.;Kajiwara, K.;Matsunaga, N.;Gonda, K.

Abstract: A flow injection analysis system was developed for the determination of ATP (free and intracellular) in micro-organisms, e.g., yeast and E. coli cultures, by a bioluminescent reaction using luciferin and Photinus-luciferin 4-monooxygenase (ATP-hydrolysing). Luminescence response was rectilinear from 1 nM to 10 µM-ATP. There was good correlation between intracellular ATP concentration. and viable cell numbers (r = 0.992). Results agreed well with those by conventional methods.
Bioluminescence Method comparison

"FIA System For Fast Glucose Measurement In Bioprocesses"
Chem. Ing. Tech. 1998 Volume 70, Issue 3 Pages 297-299
Karsten Schöngarth, Priv.-Doz. Dr. Bernd Hitzmann, Karl Friehs

Abstract: A flow injection analysis system is presented for the determination of glucose concentration. online in bioprocesses without a sampler. It is based on the injection of a glucose oxidase solution into the fluid and the determination of the O consumption in the glucose turnover. In a culture of Escherichia coli the measuring error was ≤5% compared to the off-line values. The measuring time was 47 s.
Glucose Process monitoring

"Interactions Of Non-detergent Sulfobetaines With Early Folding Intermediates Facilitate In Vitro Protein Renaturation"
Eur. J. Biochem. 1998 Volume 256, Issue 1 Pages 128-135
L Vuillard, T Rabilloud and ME Goldberg

Abstract: Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. and Rabilloud, T. (1996) Folding and Design 1, 21-27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulfonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulfide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.
Protein, folding Hen lysozyme Enzyme, tryptophan synthase Fluorescence Stopped-flow Kinetic

"Equilibrium Binding Studies Of Non-claret Disjunctional Protein (Ncd) Reveal Cooperative Interactions Between The Motor Domains"
J. Biol. Chem. 1998 Volume 273, Issue 52 Pages 35307-35318
Kelly A. Foster, John J. Correia, and Susan P. Gilbert

Abstract: Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-β, γ-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.
Binding affinity Stopped-flow Kinetic

"Online Determination Of Intracellular β-galactosidase Activity In Recombinant Escherichia Coli Using Flow Injection Analysis (FIA)"
J. Biotechnol. 1991 Volume 20, Issue 1 Pages 95-104
Heinrich-Andreas Kracke-Helm*, Lutz Brandes, Bernd Hitzmann, Ursula Rinas** and Karl Schügerl*

Abstract: A flow injection analysis (FIA) system was developed for the determination of cytoplasmic β-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for online monitoring of β-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for online monitoring of intracellular protein formation. The method is based on that described by Miller ('Experiments in Molecular Genetics', Cold Spring Harbour Laboratory, New York, USA, 1982). Cells were grown in a Luria broth in an airlift tower loop reactor (loc. cit.). The reactor was equipped with a sterile sampling device to provide a continuous sample flow for analysis of media compounds and by-products. Data acquisition and control of the flow injection system were carried out with the CASFA process management and control system (Frueh et al., Biotech-Forum, 1986, 3, 204) and the FERAS local data management system (Wieneke, Ph.D. Thesis, Univ. Hannover, 1989).
Enzyme, galactosidase Reactor Process monitoring Computer

"Control Of Microbial Activity By Flow Injection Analysis During High Cell Density Cultivation Of Escherichia Coli"
J. Biotechnol. 1993 Volume 27, Issue 2 Pages 143-157
T. Ding, U. Bilitewski*, R. D. Schmid, D. J. Korz and E. A. Sanders

Abstract: The application of an automated flow injection analysis (FIA) system for online determination of microbial activity, during high cell density cultivations of Escherichia coli is reported. Based on a bioelectrochemical principle, the FIA method used a redox mediator (potassium hexacyanoferrate(III)) to facilitate electron transfer from the microorganisms to an electrochemical detector. Assays were carried out using a new sampling device which provided aseptic operation by use of a valve and chemical sterilisation. No sample dilution or pretreatment was necessary for biomass concentrations up to approximately 40 g l-1. The sample volume was 0.5 mL and the overall analysis time was 5 min. FIA signals were found to correlate well with the oxygen uptake rate (OUR). Changes in metabolic activity due to low substrate levels or high inhibitor concentrations in the cultivation medium became obvious from the FIA signals.
Microbial activity Electrochemical analysis Process monitoring

"The Kinetic Folding Intermediate Of Ribonuclease H Resembles The Acid Molten Globule And Partially Unfolded Molecules Detected Under Native Conditions"
Nat. Struct. Biol. 1997 Volume 4, Issue 4 Pages 298-304
Tanya M. Raschke and Susan Marqusee

Abstract: Folding of ribonuclease HI from Escherichia coli populates a kinetic intermediate detectable by stopped-flow circular dichroism. Pulse labelling hydrogen exchange reveals that this intermediate consists of a structured core region of the protein, namely helices A and D and β-strand 4. This kinetic intermediate resembles both the acid molten globule of ribonuclease HI and rarely populated, partially unfolded forms detected under native conditions. These results indicate that the first portion of ribonuclease HI to fold is the most thermodynamically stable region of the native state, and that folding of this protein follows a hierarchical process.
Protein, folding Enzyme, ribonuclease hi CD Kinetic Stopped-flow

"RecA Tests Homology At Both Pairing And Strand Exchange"
Proc. Natl. Acad. Sci. USA 1997 Volume 94, Issue 22 Pages 11863-11868

Abstract: RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. To observe the effects of mismatches on the kinetics of the RecA-catalyzed recombination reaction, we used assays based upon fluorescence energy transfer that can differentiate between the pairing and strand displacement phases. Oligonucleotide sequences that produced 2-14% mismatches in the heteroduplex product of strand exchange were tested, as well as completely homologous and heterologous sequences. The equilibrium constant for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and an increase in the rate of dissociation of the intermediates. In addition, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches. The equilibrium constant for pairing and the rate constant for strand displacement both decreased 6-fold as the heterology increased to 14%. These results suggest that discrimination of homology from heterology occurs during both pairing and strand exchange. Flow Injection Analysis is used.
Protein, activity Fluorescence Kinetic

"A Rapid Enzyme Assay For ß-galactosidase Using Optically Gated Sample Introduction On A Microfabricated Chip"
Anal. Bioanal. Chem. 2004 Volume 378, Issue 7 Pages 1710-1715
Hongwei Xu, Andrew G. Ewing

Abstract: The ability to perform enzyme assays on microchips is demonstrated using optically gated sample introduction. The hydrolysis of fluorescein mono-β-d-galactopyranoside (FMG) by β-d-galactosidase (β-Gal) is continuously monitored using a microchip for 5 to 10 min. The outcome of the reaction was analyzed by performing serial on-chip separations of fluorescent substrate, FMG, and product, fluorescein. Kinetic information about β-Gal has been successfully obtained by varying the concentration of FMG. β-Gal enzymes from two different sources including bovine liver and E. coli., have been examined and compared to each other and to results obtained using traditional assay methods. In addition, the competitive inhibition of β-Gal by phenylethyl β-d-thiogalactoside (PETG) and β-lactose has been studied using this technique. PETG is found to have higher inhibition than lactose in the hydrolysis. This separation-based enzyme assay technique avoids the possible fluorescence interference between FMG and fluorescein, which is a problem with the traditional plate assay method. Additionally, the amount of the enzyme and substrate required with this technique is at least four orders of magnitude lower than the traditional plate assay method. By using optically gated sample introduction, microchips allow continuous serial injections and separations without any potential switch, thus making this technique ideal as a sensor for enzyme assays. This technique should therefore be valuable for high-throughput screening in the drug discovery industry.
Enzyme, β-d-galactosidase Galactoside conjugates Fluorescence Sensor Kinetic High throughput