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Microbial BioinsecticideBacillus thuringiensis

Dr. Aris Tri Wahyudi

Department of BiologyBogor Agricultural University

2008

Microbial Insecticide

A microbial bioinsecticide is an organism thateither produces a toxic substances that kills aninsect species or has the capability of fatallyinfecting a specific target insectThe most studied, most efective, and most oftenutilized are the toxins synthesized by Bacillusthuringiensis (Bt).Bt comprises a number of different strains(subspecies; subsp), each of which producesdifferent toxin that can kill certain specific insect

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Insecticide

Bt Toxin

, 95 %Protein, 5% CH

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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, theprotoxin is activated within its gut by the combination of alkalinepH (7.5--8.0->>) and specific digestive proteases, which convertsthe protoxin into an active toxin (Mol Mass ~68 kDa.)In its active form, the toxic protein inserts itself into themembrane of the gut epithelial cells of the insect and create anion channel through which there is believed to be an exessiveloss of cellular ATP.Cellular metabolism ceases, the insect stops feeding, becomesdehydrated, and eventually dies. Because conversion of protoxinto active toxin requires : alkaline pH and specific proteases .

Specific Activation of Bt

Ion channel

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Mode of ActionEfflux of ATP

Toxin protein

Outside cell

Gut epithelialCell membrane

Inside cell

Insertion of the B. thuringiensis toxin into the membrane of an insectGut epithelial cell. Toxin forms an ion channel between the cell cyto-plasm and the external environment

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Toxin genes are located on plasmidor chromosomal DNA?

Total cellular DNA is isolated and separated intoplasmid and chromosomal DNA by CsCl2gradient centrifugation.Protoxin gene part of genome, clone bank iscontructed from chromosomal DNA.Protoxin gene is a plasmid encoded, plasmid

DNA can be further fractionated by sucrosegradient centrifugation….separate differentplasmid 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 plasmidcarries protoxin gene?......... By Succrose GrdientThree 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 withSau3AI and ligated into BamHI site of plasmidpBR322.Transformation to E. coli, and screening by:

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Screening colonies carryingprotoxin gene

Colonies are transferred from agar plate tonitrocellulose membrane.The transferred colonies are lysed: NaOHProtoxin gene 71 kb plasmid used as a probelabelled with digoxigenin (Non--radioactive)tohybridize with plasmid (hybridization)Visualization by X--ray film

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Genetic Engineering of Bt

Expression of toxin gene is occurred only duringsporulationDNA fragment containing a toxin gene that lack itsnative promoter cloned into a plasmid under thecontrol of continupusly active, constitutive promoterfrom Tc resistant gene that had been originally isolatedfrom B. cereus plasmid and reintroduced to Bt-----Active toxin protein was produced continuouslythroughout the growth cycle: vegetative and sporulatingphases.

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Tn5

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Bt Toxin Engineering

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Bt Toxin in Water and Underfoot

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Scorpio-Toxin Baculovirus