Difference between revisions of "Part:BBa K2415000"

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Cry4Ba

Cry4Ba is an 130kDa insecticidal crystal proteins(ICP) that can be isolated from Bacillus thuringiensis subsp. israelensis (Bti). It is specifically toxic against Aedes and Anopheles mosquitoes larvae which are the vectors of Dengue virus, yellow fever virus, and malaria parasite. The Cry inclusions are solubilized in the midgut lumen of susceptible insects larvae at alkaline pH for both dipterans and lepidopterans, and proteolytically activated by gut proteases to yield the active toxins of ~65-kDa. Subsequently, the activated toxins bind specifically to a variety of receptors such as GPI (glycosylphosphatidyl inositol)-anchored aminopeptidase-N and GPI-anchored alkaline phosphatase that are found on the brush-border membrane (BBM) of midgut epithelial cells. This toxin-receptor interaction can assist toxin penetration into the cell membrane, leading to the formation of ion-permeable pores and eventually causeing osmotic lysis of target cells.

Sporulating cells of Bacillus thuringiensis (Bti) can produce insecticidal crystal proteins, or ICPs. The ICPs are classified by amino acid sequence homology into two main families, the Cry and the Cyt toxins. Cry insecticidal proteins, which are known as δ-endotoxins, are produced by the Gram-positive soil bacterium Bacillus thuringiensis (Bti) produced in the form of crystalline inclusions during sporulation. These crystal toxins are cytolytic pore-forming toxins that are lethal to various insect larvae.

The structure of Cry toxin shows a common topology composed of three domains. Domain I is an a-helix bundle, which is believed to be responsible for pore formation on the basis of its structural characteristics. Domain II is a b-prism of three antiparallel sheets with loops ending at its apex, which is thought to be involved in receptor binding. Domain III is a lectin-like b-sandwich, which is important to the stability of the toxin, and has been found to participate in determining toxin specificity.

The following picture shows the amplification of the gene cry4Ba by PCR.

The following picture shows expression and purification of Cry4Ba. Lane 1 is our negative control containing bacterial protein of E. coli BL21, lane 2 is our target protein before purification containing bacterial protein of Cry4Ba E. coli BL21, lane 3 is the protein Cry4Ba after purification. It can be seen from the graph that we have successfully obtained the target protein with high purity.

We used the purified Cry4Ba E.coli BL21 to do a toxicity test to find out the suitable concentration of killing Aedes larvae. According to the figure below, there is an obvious trend that experiment groups with higher concentrations of Cry4Ba E.coli have higher lethality rates.

Sequence and Features