본 실험에서는 Bacillus thuringiensis B-1, B-2를 배양하여 내독소 결정체와 포자를 분리하기 위해 discontinuous sucrose gradient centrifugation을 수행하였다. 분리한 내독소 단백질을 전자현미경(...
본 실험에서는 Bacillus thuringiensis B-1, B-2를 배양하여 내독소 결정체와 포자를 분리하기 위해 discontinuous sucrose gradient centrifugation을 수행하였다. 분리한 내독소 단백질을 전자현미경(SEM, TEM)을 이용하여 관찰한 결과 B. thuringiensis B-1은 길이가 1.73㎛, 폭이 0.7㎛인 이중피라미드형(bipyramidal type)을, B. thuringiensis B-2는 길이가 1.1㎛, 폭이 0.9㎛인 구형(spherical type)의 내독소 결정체를 나타내었다. B. thuringiensis B-1, B-2의 내독소 결정체 단백질의 구성을 SDS-PAGE를 통해 분석한 결과 B. thuringiensis B-1은 분자량이 약 78, 37, 28KDa의 밴드를 형성하였고, B. thuringiensis B-2는 분자량이 약 50, 35KDa의 밴드를 형성하였다. B. thuringiensis B-1, B-2의 내독소 단백질을 알칼리 완충용액(alkaline buffer)을 이용해 solubilization activity를 측정한 후, 단백질 분해효소(protease)인 trypsin을 이용하여 activation activity를 측정한 결과, B. thuringiensis B-1은 78, 37KDa 밴드는 사라지고, 28KDa인 trypsin-resistance fragment가 남아있었고, B. thuringiensis B-2는 50, 35KDa 밴드 모두가 사라지고, 분자량이 30KDa인 trypsin-resistance fragment가 새롭게 형성되었다.
본 실험에서는 Bacillus thuringiensis B-1, B-2를 배양하여 내독소 결정체와 포자를 분리하기 위해 discontinuous sucrose gradient centrifugation을 수행하였다. 분리한 내독소 단백질을 전자현미경(SEM, TEM)을 이용하여 관찰한 결과 B. thuringiensis B-1은 길이가 1.73㎛, 폭이 0.7㎛인 이중피라미드형(bipyramidal type)을, B. thuringiensis B-2는 길이가 1.1㎛, 폭이 0.9㎛인 구형(spherical type)의 내독소 결정체를 나타내었다. B. thuringiensis B-1, B-2의 내독소 결정체 단백질의 구성을 SDS-PAGE를 통해 분석한 결과 B. thuringiensis B-1은 분자량이 약 78, 37, 28KDa의 밴드를 형성하였고, B. thuringiensis B-2는 분자량이 약 50, 35KDa의 밴드를 형성하였다. B. thuringiensis B-1, B-2의 내독소 단백질을 알칼리 완충용액(alkaline buffer)을 이용해 solubilization activity를 측정한 후, 단백질 분해효소(protease)인 trypsin을 이용하여 activation activity를 측정한 결과, B. thuringiensis B-1은 78, 37KDa 밴드는 사라지고, 28KDa인 trypsin-resistance fragment가 남아있었고, B. thuringiensis B-2는 50, 35KDa 밴드 모두가 사라지고, 분자량이 30KDa인 trypsin-resistance fragment가 새롭게 형성되었다.
Bacillus thuringiensis(Bt) is a well-known species of entomo- phathogenic bacteria that is widely used as a biopesticide against many insect pests. Its produced parasporal crystals and endo- spores in their cells during sporulation. In this report, Bacillus thuringiensis B-1 and B-2 were characteriz...
Bacillus thuringiensis(Bt) is a well-known species of entomo- phathogenic bacteria that is widely used as a biopesticide against many insect pests. Its produced parasporal crystals and endo- spores in their cells during sporulation. In this report, Bacillus thuringiensis B-1 and B-2 were characterized by its ultrastruc- ture, the protein composition of its crystal proteins, activation assay of proteins by alkaline buffer and protease. B. thuringiensis B-1, B-2 cultured in the GBY medium that was high the production of δ-endotoxin, containing high amounts of nitrogen sources limiting carbohydrate sources and then their endotoxin crystals were purified with discontinuous sucrose density gradient centrifugation. The crystals of B. thuringiensis B-1 and B-2 were banded each between 65% to 70% and 70% to 75% concentrations of sucrose gradient. Morphologycally, the crystal protein from the B. thuringiensis B-1 was a bipyramidal type, whose size was 1.73㎛ x 0.7㎛, but the crystal protein from the B. thuringiensis B-2 was spherical type, whose size was 1.1㎛ x 0.9㎛, on Scanning Electron Microscope(SEM) and Transmission Electron Microscope(TEM). The crystal protein from the B. thuringiensis B-1 was composed of 78, 37, 28KDa and B. thuringiensis B-2 was composed of 50, 35KDa bands on SDS-PAGE. The crystal proteins of B. thuringiensis B-1 and B-2 solubilized in alkaline buffer(pH10) for various times. Digested B. thuringiensis B-1 and B-2 crystals showed each one fragment with a size of ca. 28KDa and 30KDa resistant to tryptic cleavage, as observed by SDS- PAGE.
Bacillus thuringiensis(Bt) is a well-known species of entomo- phathogenic bacteria that is widely used as a biopesticide against many insect pests. Its produced parasporal crystals and endo- spores in their cells during sporulation. In this report, Bacillus thuringiensis B-1 and B-2 were characterized by its ultrastruc- ture, the protein composition of its crystal proteins, activation assay of proteins by alkaline buffer and protease. B. thuringiensis B-1, B-2 cultured in the GBY medium that was high the production of δ-endotoxin, containing high amounts of nitrogen sources limiting carbohydrate sources and then their endotoxin crystals were purified with discontinuous sucrose density gradient centrifugation. The crystals of B. thuringiensis B-1 and B-2 were banded each between 65% to 70% and 70% to 75% concentrations of sucrose gradient. Morphologycally, the crystal protein from the B. thuringiensis B-1 was a bipyramidal type, whose size was 1.73㎛ x 0.7㎛, but the crystal protein from the B. thuringiensis B-2 was spherical type, whose size was 1.1㎛ x 0.9㎛, on Scanning Electron Microscope(SEM) and Transmission Electron Microscope(TEM). The crystal protein from the B. thuringiensis B-1 was composed of 78, 37, 28KDa and B. thuringiensis B-2 was composed of 50, 35KDa bands on SDS-PAGE. The crystal proteins of B. thuringiensis B-1 and B-2 solubilized in alkaline buffer(pH10) for various times. Digested B. thuringiensis B-1 and B-2 crystals showed each one fragment with a size of ca. 28KDa and 30KDa resistant to tryptic cleavage, as observed by SDS- PAGE.
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