Methodology for fast evaluation of Bacillus thuringiensis crystal protein content

Abstract

The development of the production and use of Bacillus thuringiensis in Brazil at a commercial scale faces certain difficulties, among them the establishment of efficient methodologies for the quantitation of toxic products to be commercialized. Presently, the amount of toxin is given in percentage by analyzing the samples total protein content. Such methodology however, does not measure the actual amount of active protein present in the product, since most strains express different endotoxin genes and might even produce b-toxin. Since the various types of toxins exhibit different antigenic characteristics, this work has as objective the utilization of fast immunological techniques to quantify the level of crystal protein. Crystal protein produced by a subspecies of Bacillus thuringiensis var. israelensis was purified by ultracentrifugation and utilized to immunize rabbits and to produce hiperimmune sera. Such sera were latter used to evaluate the level of proteins on commercial bioinsecticide and on laboratory cultures of B. thuringiensis through the immunodot technique. The results were obtained by comparison of data obtained from reactions with known concentrations of crystal protein permitting to evaluate the level of such protein on various materials.

Diversity and Toxicity of <i>Bacillus thuringiensis</i> from Shifting Cultivation (Jhum) Habitat

“…ANOVA analysis showed higher mortality F value -88.526, df 14, P < 0.0001 , lower lethal dose F value -5.39 x10 10 , df 14, P < 0.0001 , and lesser time to kill F value -691.87, df 14, P < 0.0001 were observed in jhum soils than Mortality at the 5 th day after the Bt application 8 10 5 cells/mL at the 5 th day after the Bt application 1,350 insects per Bt isolate Bt application 8 10 4 Bt cells/mL B-Bipyramidal; C -Cuboidal; O -Ovoidal and spherical. and 38 kDa show toxicity to different insect orders Crickmore et al, 2012;Alves and Lemos, 2000;Mahadeva Swamy et al, 2013 . Therefore, analysis of crystal proteins s profile could be useful to predict the kDa , coleopteran active -cry 3 66-73 and 70-75 kDa , dipteran-active cry 4 125-145, 68, 72, 78, 128, 130-140 ;6,Hmunpui HP7;7,Bt israelensis 4Q1 Israel;8,Khengkhawng KK1;9,Lunglei LL;10,Lengpui LP1;11,Lengpui LP2;12,Lengpui LP3;13,Lengpui LP4;14,Lengte,LT1;15,Champhai CH1;16,RihDil RD2;17,Ramrikawn RK1;18,Rawpuichhip RP1;19,Serchhip SC1;20,Seling SE1;21,Sihhmui SH1;22,Serkawr SK 1;23,Sailam SL1;24,Sailam SL2;25,Sairang SR1;26,Thenzawl TZ1;…”

“…The production of Bt in commercial scale is economically important and in order to develop its production it is necessary to determine the time to harvest. Bt strains express different endotoxin genes and crystal proteins and their harvesting time also varied from strain to strain Alves and Lemos, 2000 . OD is an indication of cell numbers in liquid media, enables the time necessary to reach stationary phase and crystal formation to be determined.…”

Immunodetection of the toxic portion of Vip3A reveals differential temporal regulation of its secretion amongBacillus thuringiensisstrains

“…After incubation, the membranes were washed three times (15 min each) with TBS‐T and incubated for 1 h with secondary antibody (anti‐IgG conjugated to alkaline phosphatase) diluted 1 : 1000 (v/v) in TBS‐T. The membranes were washed three times again and finally revealed with 1‐Step™ nitro blue tetrazolium/5‐bromo‐4‐chloro‐3‐indolyl‐phosphate (NBT/BCIP) (Alves and Lemos ).…”