Keywords:
bacteria, Bradford reagent, Coomassie Brilliant Blue G-250, Cry protein, sporulation.Abstract
The Cry proteins of Bacillus thuringiensis (Bt) produce parasporal crystal during sporulation which are toxic against insect larvae of lepidopteran, coleopteran and dipteran. In the present study, 10 native isolations of Bt strains were quantified from the Native Strains Bank of the Jorge Tadeo Lozano University, isolated from soil in the municipalities of Ráquira, Santa Sofía, Villa de Leyva and Sutamarchán, in Boyacá; and also in Susa, Cundinamarca. Crude extracts of total proteins from the 10 strains incubated in Luria-Bertani medium (LB) for 15 days at 37 °C were obtained. For quantification of total protein of the spore crystal mixture was used the Bradford method using a standard pattern a calibration curve with bovine serum albumin (BSA). After 5 minutes, the determination was realized in the spectrophotometer at a wavelength of 595 nm. The brighter blue were presented by the strains ZBUJTL35 and ZBUJTL39, with a concentration of 560.71 and 526.43 mg / ml, respectively. The Bradford method is a simple and fast technique to estimate the protein concentration. The strains will be used to determine the toxicity over Bemisiatabaci larvae.
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References
BRAVO, A. & CERÓN, J. 2004. Bacillus thuringiensis en el control biológico. ED., La Buena Semilla. Colombia. 294 pp.
BRUSSOCK, S. M., & CURRIER, T. C. 1990. Use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to quantify Bacillus thuringiensis δ-endotoxins. Analytical Chemistry. 87 pp.
FEILTELSON, J. 1993. The Bacillus thuringiensis family tree. Advanced Engineered Pesticides. 63 -71.
FERNÁNDEZ, E. & GALVÁN, A. 2010. Métodos para la cuantificación de proteínas. En: Archivos de interés: Prácticas Generales de Bioquímica y Biología Molecular. Universidad de Córdoba. Disponible en http://www.uco.es/dptos/bioquimica-biol-mol/pdfs/27%20m%c3%89todos%20para%20la%20cuantificaci%-c3%93n%20de%20prote%c3%8dnas.pdf Accesado: 28/08/2013.
JOUNG, K. B. & COTE, J.C. 2000. A review of the environmental impacts of the microbial insecticide Bacillus thuringiensis. Technicall Bulletin No. 29. Horticultural Research and Development Centre. Canada. 16 pp.
HÖFTE, H., & WHITELEY, H. 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiological reviews 53 (2): 242-255.
HÖLT, J., KRIEG, N., SNEATH, P., STALEY, J., & WILLIAMS, S. 2000. Bergey’s Manual of Determinative Bacteriology. ED., Lippincott Williams & Wilkins. 605-607.
IBRAHIM, M. A., GRIKO, N., JUNKER, M., & BULLA, L. A. 2010. Bacillus thuringiensis: a genomics and proteomics perspective. Bioeng Bugs 1 (1): 31-50.
LECADET, M., & DEDONDERT, R. 1971. Biogenesis of the crystaline inclusion of Bacillus thuringiensis during sporulation. European Journal of Biochemistry 23 (2): 282-294.
PATEIRO-LÓPEZ, B., & GONZÁLEZ-MANTEIGA, W. 2006. Multivariate partially linear models. Statistics & Probability Letters 76 (14): 1543-1549.
RAMÍREZ, L., & RAMÍREZ, N. 2009. Aislamiento y caracterización molecular y biológica de cepas nativas de Bacillus thuringiensis para el control de Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), Insecto plaga del tomate (Lycopersicum esculentum). Pontificia Universidad Javeriana. Tesis. Colombia. 184 pp.
SCHNEPF, E., CRICKMORE, N., VAN RIE, J., LERECLUS, D., BAUM, J., FEITELSON, J., & DEAN, D. H. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and molecular biology reviews 62 (3): 775-806.
SOLÍS, B. C. 2005. Cuantificación por Densitometría de la proteína Cry de Bacillus thuringiensis. Fitosanidad 9 (1): 43-46.
STOSCHECK, CM. 1990. Quantitation of Protein. Methods in Enzymology 182: 50-68.