• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.110-116
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• 2004

The over-expression in E. coli of the pHLN1-SO(＋) and pHLN2-80(－) plasmids cloned an insecticidal crystal protein (ICP) gene (crylAal type) from Bacillus thuringiensis var. kurstaki HD 1 was investigated through in part, the deletion of -80 bp promoter and an alternative change of cloning vector system. Two recombinant plasmids were constructed in an attempt to analyze the over-expression of the ICP in relations to its gene structure possessing only -14 bp ［Shine-Dalgarno (SD) sequence of -80 bp promoter］. Also, anther two recombinant plasmids similarly cloned the icp gene in a different vector system. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone, pHLRBS1-14 clone in which only the SD sequence in the inverted orientation icp gene appeared, was more evident than the pHLRBS2-14 clone in which only the -14 bp SD sequence of the right orientated icp gene was shown to exist. The pHLN2-80(－) clone produced more ICP proteins than the pHLRBS1-14 clone. In the two clones, pHLNUC1-80 right-oriented icp gene and the pHLNUC2-80 clone inverted-orientation icp gene in a new different vector, the pHLNUC2-80 produced more ICP proteins in E. coli system. These results indicate that the P/ac promoter, the inverted icp gene insertion and -80 bp promoter (-66 bp part of the icp gene promoters), were concerned with the expression of the icp gene in the recombinant plasmids. In addition, the expression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.497-506
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• 1998

The expression in Escherichia coli of a cloned insecticidal protein (ICP) gene from Bacillus thuringiensis var. kurstaki HD1 in pHLN1-80 (+) and pHLN2-80(-) plasmids was investigated through deletions in promoters, transcription start point, and termination region. Six recombinant plasmids were constructed in an attempt to analyze the overexpression of the ICP in relations to its gene structure. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone was not overexpressed which having only -80 bp (contained BtI promoter) part of the ICP gene promoter (without Plac promoter), the right-oriented ICP gene and the termination region. Removal of 350 bp from upstream region of the Plac of the clone pHLN2-80 (-) resulted in overexpression of the ICP. One clone was not overexpressed in which the clone consisted of -72 bp part of the ICP promoter without the transcription start point and the transcriptional termination region, and having the right-oriented ICP gene sequence. One clone consisting of the inverted ICP gene sequence, the -72 bp ICP gene promoter, and without the termination region caused overexpression. One clone which consisted of the inverted ICP gene, the -72 bp ICP gene promoter and the termination sequence was overexpressed. These results indicated that the Plac promoter, transcription termination region, the inverted ICP gene insertion, and the -80 bp or -72 bp part of the ICP gene promoters were concerned in the overexpression of the ICP gene in the recombinant plasmid, and also the overexpression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.

• Applied Biological Chemistry
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• v.22 no.2
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• pp.123-134
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• 1979

For the purpose of developing a microbial insecticide utilizing Bacillus thuringiensis Berliner, research was done and the following results were obtained. 1) As the freeze-dried matter of the cocoon-cooked water discarded from the filature contains much crude protein(51.825%) and a lot of inorganic salts, it can make a good nutrition source for the culture cf B. thuringiensis Berliner. 2) Based on the suspensibility, formula F-5 turned out to be the most suitable for insecticidal use. Its composition includes 0.2 g of the cell-spore-crystal mixture, 25 g of 200-mesh kaolin, 2.5 g of New Kalgen-NX-150, and 2.5 g of glycerine admixed with 8 ml of distilled water and granulated in 80-mesh size. 3) All the components of F-5, F-6 and F-7 are identical except that the amounts of cell-spore-crystal mixture of F-5, F-6, and F-7 are 0.2 g, 0.4 g, and 0.6 g, respectively. Accordingly, their physical properties are almost all the same. 4) Formulas F-5, F-6, and F-7 exhibited an excellent toxicity to Anomis mesogona Walker, Dendrolimus spectabilis Butler, and Margaronia perspectalis Walker at the concentration of 5%. 5) Formulas F-8 and F-9 which contain $NaHCO_3$ as one of their components showed a remarkably reduced toxicity to Anomis mesogona Walker and Dendrolimus spectabilis Butler than F-6 which does not contain $NaHCO_3$. 6) A maximum of $2.97{\times}10^9$ spores per ml was obtained by incubating B. thuringiensis in M-3 which has a pH of 7.05 and comprises 0.2% of ammonium sulphate and 0.8% of glucose dissolved in the cocoon-cooked water, with aeration for 96 hours. 7) Formula F-6 exhibited a somewhat reduced toxicity to Anomis mesogona Walker and Dendrolimus spectabilis Butler, when stored at room temperature for 70 days after formulation and it is desirable to keep it in a dark and cold place. 8) In held applications, formula F-6 showed a good activity in controlling Monema flavescens Walker. Margaronia perspectalis Walker, and Macrosiphum ibarae Matsumura.