1 9/21/2010 Microbial Bioinsecticide Bacillus thuringiensis Dr. Aris Tri Wahyudi Department of Biology Bogor Agricultural University 2008 Microbial Insecticide A microbial bioinsecticide is an organism that either produces a toxic substances that kills an insect species or has the capability of fatally infecting a specific target insect The most studied, most efective, and most often utilized are the toxins synthesized by Bacillus thuringiensis (Bt). Bt comprises a number of different strains (subspecies; subsp), each of which produces different toxin that can kill certain specific insect

9/21/ Insecticide Bt Toxin, 95 %Protein, 5% CH

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7 Mode of Action The parasporal crystal is not the active form of the insecticide, rather, it is a protoxin, a precursor of the active toxin. When the parasporal crystal is ingested by a target insect, the protoxin is activated within its gut by the combination of alkaline pH ( >>) and specific digestive proteases, which converts the protoxin into an active toxin (Mol Mass ~68 kDa.) In its active form, the toxic protein inserts itself into the membrane of the gut epithelial cells of the insect and create an ion channel through which there is believed to be an exessive loss of cellular ATP. Cellular metabolism ceases, the insect stops feeding, becomes dehydrated, and eventually dies. Because conversion of protoxin to active toxin requires : alkaline pH and specific proteases. Specific Activation of Bt

Ion channel 9/21/2010 Mode of Action Efflux of ATP Toxin protein Outside cell Gut epithelial Cell membrane Inside cell Insertion of the B. thuringiensis toxin into the membrane of an insect Gut epithelial cell. Toxin forms an ion channel between the cell cyto- plasm and the external environment 8

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12 9/21/2010 Toxin genes are located on plasmid or chromosomal DNA? Total cellular DNA is isolated and separated into plasmid and chromosomal DNA by CsCl2 gradient centrifugation. Protoxin gene part of genome, clone bank is contructed from chromosomal DNA. Protoxin gene is a plasmid encoded, plasmid DNA can be further fractionated by sucrose gradient centrifugation….separate different plasmid according to their size. B. thuringiensis subsp kurstakii Protoxin gene on one of 7 different plasmid: 2.0, 7.4, 7.8, 8.2, 14.4, 45, and 71 kb. Which plasmid carries protoxin gene? By Succrose Grdient Three fractions: small (2 kb); medium (7.4, 7.8, 8.2, 14.4 kb); large (45, and 71 kb). Medium and large are partially digested with Sau3AI and ligated into BamHI site of plasmid pBR322. Transformation to E. coli, and screening by:

13 9/21/2010 Screening colonies carrying protoxin gene Colonies are transferred from agar plate to nitrocellulose membrane. The transferred colonies are lysed: NaOH Protoxin gene 71 kb plasmid used as a probe labelled with digoxigenin (Non--radioactive)to hybridize with plasmid (hybridization) Visualization by X--ray film

14 9/21/2010 Genetic Engineering of Bt Expression of toxin gene is occurred only during sporulation DNA fragment containing a toxin gene that lack its native promoter cloned into a plasmid under the control of continupusly active, constitutive promoter from Tc resistant gene that had been originally isolated from B. cereus plasmid and reintroduced to Bt----- Active toxin protein was produced continuously throughout the growth cycle: vegetative and sporulating phases.

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9/21/ Bt Toxin Engineering

9/21/ Bt Toxin in Water and Underfoot

9/21/ Scorpio-Toxin Baculovirus