Team:GZHS-United/Design
Design
Our project is to make a new biological mosquito killer to kill mosquitoes in an environmental friendly way. There are mainly two active components in our product: protein Cry11Aa and recombinant Aedes aegypti densoviruses. We mixed them together to make effective and environmental friendly mosquito killer. Our product shows a high specificity for mosquitoes as a host. It is relatively stable in the environment and have the potential to spread and persist in mosquito populations.
How do they work?
Cry proteins
Protein Cry11Aa is produced as protoxins that are solubilized in the mosquito midgut lumen due to its characteristic pH and reducing conditions. The soluble protoxins are then activated by midgut proteases to release the toxin fragment. The Cry toxic fragment binds to specific receptors located in the microvilli of the apical membrane of midgut epithelial cells. Oligomerization of the toxin that follows leads to membrane insertion, pore formation and cell lysis [1].
BmK IT1
BmK IT1 is an excitatory insect-specific toxin from Buthus martensii Karsch, and it is an insect-selective single-chain neurotoxic polypeptide that is composed of 88 amino acid residues cross-linked by four disulfide bridges. It has been shown to affect insect neuronal sodium conductance by binding to excitable sodium channels [2].
Adedes aegypti densoviruses
Adedes aegypti densoviruses are small, autonomous, non-enveloped DNA viruses with small single-stranded DNA genomes. These viruses are pathogenic to their hosts, they replicate in the nuclei of cells of mosquitoes and cause characteristic histopathological signs, which include hypertrophied, densely stained nuclei [3].
Protein design
Our project is consist of two parts: the protein part and the virus part.
This is an overview of our protein part . First of all, we extracted genome of Bt.i, and then use PCR to amplify the target gene Cry11Aa. These target sequences were then assembled into the expression vector pEASY-Blunt and then transformed into E.coli BL21 (DE3). The expressed protein was extracted after cell lysis.
That is the expression vector. It is controlled by T7 promoter and regulated by LacI and LacO. The expression of the two target proteins CryAa could be induced by IPTG, and both of them would be purified using nickel column due to the his-tag.
Virus design
pUCA
The plasmid pUCA contains the genome of Adedes aegypti densoviruses in pUC19.
p7NS1-GFP
The plasmid p7NS1-GFP is mostly similar to the plasmid pUCA. The difference is that the segment of gene which codes the structural protein VP is replaced by GFP.
NS1-BIT-GFP
When we insert the our target gene BmK IT1 into p7NS1-GFP, NS1-BIT-GFP was formed.
This is an overview of our virus part. First, we transformed our initial plasmids pUCA and p7NS1-GFP, respectively, into E.coli DH5α to culture in large scale, and we extracted them after some time. Then, we assembled our target gene BmK IT into p7NS1-GFP and we will get the plasmid NS1-BIT-GFP. After that, we cotranfected plasmids NS1-BIT-GFP and pUCA into Aedes albopictus C6/36 cells (ATCC CRL-1660). Recombinant viruses were then produced by plasmid NS1-BIT-GFP, with the helper plasmid pUCA.
Reference
[1] Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produce
[2] An overview of toxins and genes from the venom of the Asian scorpion Buthus martensii Karsch
[3] A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus
