Characterization and selection of Bacillus thuringiensis isolates effective against Sitophilus oryzae

The entomopathogenic bacterium Bacillus thuringiensis is a control agent with toxic and environmental characteristics that allows the control of pest insects according to the Integrate Pest Management (IPM) precepts. In order to find new strains, potentially toxic to Sitophilus oryzae L. 1763 (Coleoptera: Curculinidae), 1.073 strains of B. thuringiensis from parts of Brazil were used. Genetic material was extracted with InstaGene Matrix kit, used for the amplification of sequences in Polymerase chain reaction (PCR), and viewed in 1.5% agarose gel. The gene cry35Ba class was represented by 60 B. thuringiensis isolates (5.6%), which were then subjected to bioassays with S. oryzae larvae. Among the isolates studied, four caused more than 50% mortality in pathogenicity tests, and the isolates 544 and 622 were the most virulent, as determined by CL50 estimates. The four toxic isolates had spherical, bi-pyramidal and cuboid crystals, and a 44-kDa protein was found in sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE), which coded for the product of cry35Ba genes. These data demonstrate the potential of B. thuringiensis for the management of S. oryzae larvae.

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Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa Synergism

Applied and Environmental Microbiology ◽

10.1128/aem.00409-09 ◽

2009 ◽

Vol 75 (14) ◽

pp. 4661-4667 ◽

Cited By ~ 26

Author(s):

Alejandro Hernández-Soto ◽

M. Cristina Del Rincón-Castro ◽

Ana M. Espinoza ◽

Jorge E. Ibarra

Keyword(s):

Bacillus Thuringiensis ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Microbial Control ◽

Control Agent ◽

Mass Spectrometry Analysis ◽

Parasporal Crystal ◽

Spectrometry Analysis ◽

Crystal Complex

ABSTRACT Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

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New Strategy for Isolating Novel Nematicidal Crystal Protein Genes from Bacillus thuringiensis Strain YBT-1518

Applied and Environmental Microbiology ◽

10.1128/aem.01346-08 ◽

2008 ◽

Vol 74 (22) ◽

pp. 6997-7001 ◽

Cited By ~ 60

Author(s):

Suxia Guo ◽

Mei Liu ◽

Donghai Peng ◽

Sisi Ji ◽

Pengxia Wang ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Meloidogyne Hapla ◽

Microscopy Observation ◽

Root Knot Nematode ◽

Dna Library ◽

Sds Page ◽

New Strategy ◽

Key Steps

ABSTRACT We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes—cry55Aa1, cry6Aa2, and cry5Ba2—were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.

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Use of Seed Protein Profiles to Characterize Peanut Cultivars1

Peanut Science ◽

10.3146/i0095-3679-21-2-18 ◽

1994 ◽

Vol 21 (2) ◽

pp. 152-159 ◽

Cited By ~ 6

Author(s):

C. M. Bianchi-Hall ◽

R. D. Keys ◽

H. T. Stalker

Keyword(s):

Seed Protein ◽

Cultivar Identification ◽

Storage Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Genetic Material ◽

Seed Storage Proteins ◽

Molecular Techniques ◽

Protein Profiles ◽

Sds Page

Abstract In the last 10 to 15 yr, the development of biotechnology and molecular techniques has allowed great advancements toward the identification of cultivars among plant species. In legumes, the success of cultivar identification depends on the species under investigation, the type and variability of genetic material found in cultivars, and the technology used for investigations. In this study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to assess diversity of peanut (Arachis hypogaea L.) seed protein profiles. The objectives of this investigation were a) to assess diversity of protein profiles in peanuts for cultivar identification using SDS-PAGE and b) to determine the extent of variability of seed storage proteins (SSP) among samples of cultivars originating from different locations. The first study included 34 cultivars grown at Lewiston, NC and the second one included nine cultivars grown at six locations. The results of both studies indicated that it is possible to differentiate between subspecies but not to associate a particular profile with only one specific cultivar. Within subspecies, cultivars clustered in more than one group and most cultivars that grouped together were genetically related.

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Seleção de cepas de Bacillus thuringensis Berliner para o controle de Aedes aegypti Linnaeus

Journal of Biotechnology and Biodiversity ◽

10.20873/jbb.uft.cemaf.v4n1.melo ◽

2013 ◽

Vol 4 (1) ◽

pp. 78-83

Author(s):

André Luiz de Almeida Melo ◽

André Luís Lopes da Silva ◽

Magda Clara Vieira da Costa Ribeiro ◽

Mitiyo Fukuda Miyaoka ◽

Vanete Thomas Soccol ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Aedes Aegypti ◽

Mosquito Species ◽

Larval Mortality ◽

Protein Profile ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sds Page ◽

Bacillus Thuringensis ◽

Two Stages

The present study aimed to select strains of Bacillus thuringiensis with insecticidal activity against Aedes aegypti. It was tested sixteen strains of bacteria, isolated from Paraná state, Brazil, that were used in laboratory assays with mosquito larvae. Tests were carried out in two stages, first one to select the most efficient strains (screening) and second to estimate LC50. The protein profile of the highest toxicity of strain was obtained by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The best performance of larval mortality was produced by BR-01 strain, which 96.7% mortality rate, significantly higher than others. In the second part, there was obtained a LC50 of 9.07 µL.L-1 fermented extract. The protein profile revealed many peptides between 15 and 140kDa, similar to that reported in Bacillus thuringiensis ser. israelensis strains which high toxicity to mosquito species.

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Crystalline protein profiling and cry gene detection in Bacillus thuringiensis strains isolated during epizootics in Cydia pomonella L.

Biological Letters ◽

10.1515/biolet-2015-0008 ◽

2014 ◽

Vol 51 (2) ◽

pp. 83-92 ◽

Cited By ~ 2

Author(s):

Edyta Konecka ◽

Jakub Baranek ◽

Adam Kaznowski

Keyword(s):

Bacillus Thuringiensis ◽

Polyacrylamide Gel Electrophoresis ◽

Cydia Pomonella ◽

Sodium Dodecyl ◽

Natural Populations ◽

Protein Profiling ◽

Gene Detection ◽

Sds Page ◽

Molecular Masses ◽

Reference Strains

AbstractThe composition of Bacillus thuringiensis crystalline inclusions was characterized in 18 strains: 12 isolates were obtained from the intestinal tract of Cydia pomonella larvae during epizootics, 2 isolates were cultured from Leucoma salicis larvae taken from their natural populations, and 4 reference strains. The number and molecular mass of B. thuringiensis crystalline proteins (Cry and Cyt) was estimated by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The crystals contained 1-8 proteins with molecular masses of 36-155 kDa. The toxin profiles differed both quantatively and qualitatively. The B. thuringiensis MPU B9 isolate had the highest number and diversity of Cry toxins. The analysis of crystal composition by SDS-PAGE was insufficient to detect groups and subgroups of Cry proteins. We identified 20 groups and 3 subgroups of Cry and Cyt crystalline toxins. Only one epizootic strain harboured cry25. In single reference strains, the cry1H, cry10 and cry25 genes were found. We did not find any correlation between the occurrence of cry genes and electrophoretic protein profiles of crystalline toxins.

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Comparison and Validation of Methods To Quantify Cry1Ab Toxin from Bacillus thuringiensis for Standardization of Insect Bioassays

Applied and Environmental Microbiology ◽

10.1128/aem.01855-07 ◽

2007 ◽

Vol 74 (1) ◽

pp. 130-135 ◽

Cited By ~ 42

Author(s):

André L. B. Crespo ◽

Terence A. Spencer ◽

Emily Nekl ◽

Marianne Pusztai-Carey ◽

William J. Moar ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Total Protein ◽

European Corn Borer ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Data Consistency ◽

Enzyme Linked Immunosorbent Assay ◽

Corn Borer ◽

Sds Page ◽

Bradford Assay

ABSTRACT Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC50 values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.

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Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646313 ◽

1992 ◽

Vol 68 (05) ◽

pp. 534-538 ◽

Cited By ~ 5

Author(s):

Nobuhiko Yoshida ◽

Shingi Imaoka ◽

Hajime Hirata ◽

Michio Matsuda ◽

Shinji Asakura

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl Sulfate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Tetraacetic Acid ◽

Fibrin Monomer ◽

Sds Page ◽

Thrombotic Tendency ◽

Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

YARSI Medical Journal ◽

10.33476/jky.v26i2.394 ◽

2018 ◽

Vol 26 (2) ◽

pp. 058

Author(s):

Anna P. Roswiem ◽

Triayu Septiani

Keyword(s):

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Sds Page

Bahan baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.

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Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200213104224 ◽

2020 ◽

Vol 20 (8) ◽

pp. 970-981

Author(s):

Hamed A. Ghramh ◽

Essam H. Ibrahim ◽

Mona Kilnay

Keyword(s):

Anticancer Activity ◽

Cancer Cells ◽

Biological Activities ◽

Sugar Content ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Bioactive Molecules ◽

Sds Page ◽

Splenic Cells ◽

Juniperus Procera

Background: Juniperus procera and Majra honey are well-known as a folk medicine in many countries. Objectives: This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Methods: Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. Results: The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells’ growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Conclusion: Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

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Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00125-2 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Preeti Anand ◽

Jay Prakash Pandey ◽

Dev Mani Pandey

Keyword(s):

San Francisco ◽

Tertiary Structure ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Evolutionary Genetics ◽

Imaging Analysis ◽

Silk Cocoon ◽

Natural Color ◽

Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

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