Investigation of Transient Expression System in Nicotiana bethamiana to Produce Novel TALEs and the Development of (ds)DNA Detection with Quantum Dot-Labeled Proteins in Graphene Oxide Quenching Arrays

Our objective was to develop a faster method of quantitively detecting double stranded (ds)DNA of pathogenic bacteria such as the Shiga Toxin 2 gene present in E. coli O157. Transcription Activator-Like Effectors (TALEs) are a new class of DNA-binding proteins which selectively bind to dsDNA with the 12th and 13th amino acids of each repeat, called repeat variable diresidues (RVDs). Novel TALE proteins were designed to target the stx2 gene and were cloned into existing AvrBs3 TALE protein in the pMAL c2x vector system for bacterial BL-21 E. Coli expression. The protein’s DNA-binding region was then subcloned pEAQ vectors for expression in N. Benthamiana expression systems. Protein expression was comparable between systems, as plant expression had a higher yield at the cost of additional time and resources. After expression, TALE proteins were purified using affinity chromatography and characterized. TALE proteins were then labeled with CdSe/ZnS Quantum Dots (QDs) using EDC and NHS labeling method to create a stable peptide bond between the QD and protein. Labeling efficiency was very high ~90% with relatively low initial protein concentration. Labeled proteins were then used in Graphene oxide (GO) quenching and sensitivity arrays to observe if GO could quench QD signal and if target DNA could restore QD signal. In both experiments, QD were excited with 300 nm light and 515 nm endpoint fluorescence data was collected. Optimal sensitivity array conditions were determined to be 10 nM protein and 2 μg/ml GO, and DNA sensitivity arrays could be completed in under 30 minutes. It is important to further investigate the binding capabilities of the TALE proteins and the sensitivity of this system in the presence on nontarget DNA and complex biosamples

Nieuwe inzichten in Xanthomonasziekte in Anthurium

Het lijkt niet duidelijk waar besmettingen met Xanthomonas axonopodis pathovar dieffenbachiae (vroeger X. campestris pathovar dieffenbachiae geheten) in de Anthuriumteelt vandaan komen. Het kan met het plantmateriaal meekomen, overleven binnen foto: sterk vergroot huidmondje of buiten de kas, of latent (zonder symptomen) aanwezig zijn. v ol Xanthomonas cellen Goede, preventieve maatregelen worden aangegeven door Anthura in enkele mailings, waarin ook de symptomen worden beschreven. PPO heeft meer informatie boven tafel gehaald uit de wetenschappelijke literatuur en uit onderzoeksgegevens van andere teelten die door Xanthomonas worden belaagd, onder meer in de bollenteelt (hyacint)

FLOW SYSTEMS FOR CLINICAL ANALYSIS USING IMMOBILIZED ENZYMES AND CHEMILUMINESCENCE DETECTION (CHROMIUM, GLUCOSE)

Analytical systems used in a clinical setting should offer high sample throughput and eae of operation, while at the same time providing selective, sensitive, accurate and precise determinations of clinically important analytes. Described in this study are two flow systems based on chemiluminescence (CL) detection that provide the features important in a practical clinical method: a flow injection analysis (FIA) system and a two phase flow system. Both systems take advantage of the specificity of enzyme reactions. FIA systems for the determination of Cr(III) and glucose, based on luminol CL were developed. The Cr(III) system used EDTA as a masking agent to eliminate interferences from other metal ions, making it selective for Cr(III). The FIA system for glucose determination in biological fluids was based on the use of glucose oxidase, which was covalently immobilized to controlled porosity glass beads. The immobilized enzymes were packed into a column incorporated into the FIA system. Optimum operating conditions, detection limits and linear ranges were determined. The system was capable of sampling rates as high as 240 samples/hour. There was no significant differences at the 95% confidence level between CL-FIA results for blood glucose determination and results obtained by a commercially available reference method. The immobilized glucose oxidase was quite stable and could be reused for many assays. The two phase flow system work employed a custom-designed flow cell that featured enzymes immobilized by entrapment by a membrane. Analyte in the flow stream could diffuse under a concentration gradient across the membrane into the reagent cell. Theory describing the performance of the cell was developed. The system was characterized using the peroxidase-catalyzed luminol reaction, firefly bioluminescence (BL) and bacterial BL. The kinetics of the luminol and firefly reactions were varied in order to observe the effect of reaction rate on response time. Rise and fall times to steady state emission were measured, as were the relative intensities for the various reaction conditions. Rise times as fast as 40 seconds were observed for the firefly reaction when 10 mg/mL of apyrase was added to the system. The bacterial BL system exhibited much slower response than the modified firefly systems, but the bacterial system was more stable over time. Response time in the bacterial system was a function of NADH concentration. The performance of these systems for ATP and NADH determination was investigated, and approximate detection limits and linear ranges were determined

Etude de la variabilité d'un agent pathogène, Xanthomonas campestris pv manihotis, par l'analyse factorielle multiple

International audienc

List of Department of Agriculture publications

THE following publications are available on application from the Department of Agriculture. Most of the bulletins listed are reprints from the Journal of Agriculture and are free of charge

Control of foliar diseases of groundnut using inorganic and metal salts

Late leaf spot (LLS) caused by Phaeoisariopsis personata and rust caused by Puccinia arachidis are the two major foliar diseases of groundnut (Arachis hypogaea) which usually occur together and significantly reduce the crop yield (Subrahmanyam et al. 1995). Desirable levels of host plant resistance against these two diseases are not available in cultivars commonly grown by farmers. Fungicidal control of LLS and rust is expensive and requires alternative strategies for management of these destructive diseases. Several inorganic and metal salts possess antifungal activity and control pre-harvest and postharvest diseases caused by pathogenic fungi (Reuveni et al. 1995, Olivier et al. 1998). These chemicals were also proved to elicit the plant defense mechanisms against invasion of pathogenic fungi (Gamil 1995). We studied the effects of 33 inorganic and metal salts on the germination of conidia of P.personata and urediniosporcs of P. arachidis under in vitro conditions and the efficacy of selected salts to reduce the incidence of LLS and rust in detached leaf bioassay

Portable light detectors for bioluminescence biosensing applications: A comprehensive review from the analytical chemist's perspective

Bioluminescence, that is the emission of light in living organisms, has been extensively explored and applied for diverse bioanalytical applications, spanning from molecular imaging to biosensing. The unprecedented technological evolution of portable light detectors opened new possibilities to implement bioluminescence detection into miniaturized devices. We are witnessing a number of applications, including DNA sequencing, reporter gene assays, DNA amplification for point-of care and point-of need analyses relying on BL. Several photon detectors are currently available for measuring low light emission, such as photomultiplier tubes (PMT), charge-coupled devices (CCD), complementary metal oxide semiconductors (CMOS), single photon avalanche diodes (SPADs), silicon photomultipliers (SiPMs) and smartphone-integrated CMOS. Each technology has pros and cons and several issues, such as temperature dependence of the instrumental specific noise, the power supply, imaging capability and ease of integration, should be considered in the selection of the most appropriate detector for the selected BL application

CLINICAL APPLICATIONS OF CHEMILUMINESCENCE DETECTION

This dissertation evaluates the potential of chemiluminescence analysis for two types of clinical applications: (1) rapid analysis with a flow injection system which consumes small reagent volumes (25 (mu)L) and (2) a new approach to chemiluminescence immunoassay. The first application studied uses the firefly reaction with a flow system to analyze for adenosine triphosphate (ATP). Two flow configurations called the valve within a valve and merging zones configurations were evaluated with respect to effect of parameters such as flow and sample volume on precision, sensitivity and sample throughput. With the appropriate choice of parameters, both systems achieve precisions of 1-2% relative standard deviation and throughputs of 5-10 measurements per minute. The merging zones configuration consumes smaller volumes of sample. The second project demonstrates a new approach to immunoassay based on chemical excitation of fluorophor labelled immunochemicals using the peroxyoxalate reaction. The effects of fluorophors, esters, and solvents, etc. on the chemiluminescence measurement were evaluated. The conditions for achieving the best precision (1-3% R.S.D.) with a high water component in the measurement were rhodamine B in Tris buffer adjusted to pH 8.00, 10(\u27-3) M bis(2,4,6-trichlorophenyl) oxalate (TCPO) in ethyl acetate and 10(\u27-3) M hydrogen peroxide in acetonitrile in the ratio 5:2:3. Under these conditions, the detection limit for rhodamine B is 10(\u27-9) M. The detection limit is established by variations in the background chemiluminescence. The possibility of both homogeneous and heterogeneous immunoassays was evaluated. It was shown that the chemiluminescence excitation efficiency decreases when fluorophors were bound to albumin but increases when the fluorophor was coupled to folic acid. The feasibility of a homogeneous immunoassay was demonstrated by binding albumin to rhodamine labelled antiserum. It was shown that the chemiluminescence intensity of the fluorophor labelled antibody decreases with increasing concentrations of bound antigen. The heterogeneous chemiluminescence immunoassay was evaluated by comparison with a commercially available fluorescence immunoassay kit by Bio Rad. Both immunoassays are limited by imprecision of mixing of the solvent systems for the chemiluminescence measurement

Daños causados por Xanthomonas campestris pv. phaseoli y su efecto en el rendimiento del frijol común (habichuela; Phaseolus vulgaris)

Common bacterial blight produced by Xanthomonas campestris pv. phaseoli (Xcp), is considered one of the diseases of major economic importance in bean (Phaseolus vulgaris) production in Honduras. Yield reduction at farm level produced by this pathogen is considered significant; however, its magnitude is unknown. To determine the severity, incidence and percentage of seed yield reduction caused by Xcp, two experiments were conducted in El Zamorano Valley, Honduras, during the early (June to Aug.) and late (Sept. to Dec.) growing seasons of 1989. Three genotypes differing ¡n their reaction to Xcp, XAN 155 (resistant), EAP 10-88 (moderately resistant) and "Catrachita" (susceptible), were evaluated under protected and unprotected treatments applied to plants artificially inoculated with Xcp. Reaction to Xcp clearly reflected the degree of resistance of each genotype. Yield losses caused by Xcp during the late season ranged between 22 and 41.6% in the unprotected treatment. The severity of Xcp observed in the resistant genotype XAN 155 suggested that at farm level the implementation of an integrated control program is necessary in order to reduce yield losses caused by this pathogen.La bacteriosis común, causada por Xanthomonas campestris pv. phaseoli (Smith) Dye (Xcp), está considerada como una de las enfermedades de mayor importancia económica en la producción de frijol (Phaseolus vulgaris) en Honduras. Las pérdidas en el rendimiento a nivel de campo causadas por el ataque de este patógeno se consideran significativas; sin embargo, no se sabe su magnitud. Para determinar la severidad, incidencia y el porcentaje de reducción en el rendimiento de grano causados por Xcp, se efectuaron dos experimentos en el Valle de El Zamorano, Honduras, en siembras durante las épocas de primera (Jun- Ago) y postrera (Sep-Dic) de 1989. Se utilizaron tres genotipos con diferencias en su reacción al ataque de Xcp, XAN 155 (resistente), EAP 10-88 (moderado) y Catrachita (susceptible), se evaluaron bajo tratamientos con protección e inoculación artificial con Xcp. La reacción a Xcp reflejó claramente la intensidad de resistencia de cada uno de los genotipos. Las pérdidas en rendimiento causadas por Xcp durante la época postrera, estuvieron entre el 22 y el 41.6% en el tratamiento con inoculación artificial. La incidencia de Xcp observada en el genotipo resistente XAN 155, sugiere que a nivel de finca es necesaria la implantación de programas de manejo integrado para reducir las pérdidas en rendimiento causadas por este patógeno