1,720,971 research outputs found
Production and electrophoresis-based purification of recombinant green fluorescent protein from Escherichia coli
Full text linkGreen fluorescent protein (GFP) can be used as markers in numerous bioscience applications due to its visible fluorescence. Availability of a reliable and affordable GFP quantitation device is important for these applications. GFP can be quantitated with a spectrofluorometer, but the accuracy of spectrofluorometric method is affected by the present of biomass and it cannot be used to differentiate the nativity of a GFP. Therefore, one of the objectives of this study was to develop a GFP quantitation method using gel-based imaging analysis. The precision, detection limit, linearity, reproducibility and accuracy of this quantitation method were investigated to demonstrate its reliability and suitability for practical application. Besides, the sensitivity and GFP nativity differentiation test were performed and compared with those obtained with a spectrofluorometer. The cultivation conditions for the GFP production in Escherichia coli BL21(DE3) were optimized using statistical experimental designs. The process variables include agitation rate, temperature, pH of medium, inducer concentration, induction time and inoculum density were screened using the fractional factorial design. The screening step identified that agitation rate, temperature and cell density for protein expression have significant effects on GFP production. Hence, these process variables were further optimized using the Box-Behnken design. The optimal cultivation conditions were 206 rpm agitation rate at 31°C and cell density (OD600 nm) of 1.04 for protein expression. The predicted GFP yield (0.234 g/L) is corresponded well with that obtained experimentally (0.241 g/L). The GFP yield achieved under the optimized conditions is 9-folds higher than that of the unoptimized conditions (0.025 g/L). A similar protein yield was achieved when these optimized conditions were applied in a 2-liter stirred tank bioreactor fermentation. Cell disruption is an important step in intracellular protein purification. However, this additional step resulted in a substantial protein loss and increased the overall processing time. Hence, a direct purification method of recombinant GFP from intact E. coli cells was developed using a preparative native polyacrylamide gel electrophoresis. This direct purification process has eliminated the cell disruption step. The cellular content of E. coli was drifted out from cells by the electrophoretic force. The GFP was further separated from other intracellular proteins through a gel and recovered by electrophoretic elution. The effects of the operating parameters including the feedstock volume, feedstock concentration, concentration of resolving gel and height of resolving gel on the purity and yield of GFP were further investigated. At 100 µL feedstock volume, 15% (w/v) feedstock concentration, 12% (w/v) resolving gel concentration and 2 cm resolving gel height, the purity and yield of GFP achieved were 98 and 88%, respectively. The scale-up study has demonstrated that the ratio of feedstock volume to column cross-sectional area at optimal ratio of 44 µL/cm2 was an important factor for scaling-up the purification process. The present study focused on gel electrophoresis technique, which provides the efficient ways to measure the amount of GFP and to purify the intracellular GFP. Besides, an optimal GFP cultivation condition was identified by using statistical experimental designs for maximum GFP production
Determination of Green Fluorescent Protein by Ultraviolet-Excited Gel Imaging
Full text linkThe green fluorescent protein has gained interest in bioanalytical applications due to its visible fluorescence. As the usage of green fluorescent protein increases, more appropriate fluorescence instrumentation is required. Most fluorescence instrumentation uses ultraviolet light as the excitation source for the determination of green fluorescent protein. However, ultraviolet radiation may damage biological molecules and affect the quantitative analysis. In this study, the effects of the ultraviolet radiation period and the mass of green fluorescent protein on the fluorescence determination were characterized using gel imaging. The ultraviolet illumination period affected the green fluorescent protein fluorescence intensity. The fluorescence increased with the ultraviolet illumination time from 30–90 s. However, the fluorescence intensity decreased when the excitation period was longer, probably due to photobleaching. The photobleaching decreased when a higher concentration of enhanced green fluorescent protein was employed. This gel imaging study has provided a better understanding of the optimum conditions for the determination of green fluorescent protein
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Seawater corrosion resistant heat transfer agent (HTA) to improve water evaporation in solar still
Full text linkSolar still is an economical technique for water desalination, however its main drawback is low productivity. In this project, a heat transfer agent (HTA) was installed in a solar still aimed to distribute the heat throughout the seawater and increase the water evaporation rate. Copper was selected as the HTA due to its high electrical and thermal conductivities. However, seawater may cause copper corrosion; hence coating is required to avoid the metal deterioration. The present study reported the effects of coating material and thickness, and the incubation temperature on the copper corrosion rate and heat transfer rate. Copper rods coated with different coating material have low weight loss and corrosion rate if compared with bare copper rod. Besides, weight loss and corrosion rate with incubation temperature of 25°C were relatively high if compared with incubation temperature of 60°C. Moreover, an increase in the coating layer linearly increased the mass of the coated copper rods. Under the steady-state condition, the thermal conductivities increased when the coating layer was increased further. Hence, adding the coating layer reduced the radial heat transfer rate and higher axial heat transfer rates were obtained. A solar still was designed and fabricated. HTA consists of a sheet of copper plate and 12 pieces of copper rods. Two set of experiment set-up were conducted to investigate the effect of HTA on the solar still performance. In general, water and glass temperatures and evaporative heat transfer of solar still with HTA were higher than the solar still without HTA. Experiment data have revealed the function of the HTA that may quickly transferred the radiation energy from the copper plate and rods to the water. The introduction of the HTA has successfully increased 30% of water evaporation rate and 18% of thermal efficiency
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