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Design

The principles of Mtx1 and Cry4Ba to kill mosquito larvae

Mtx1

The mature MTX (without the putative signal sequence) is processed into a 27 kDa N-terminal fragment and a 70 kDa C-terminal fragment by trypsin. The N-terminal ADP ribosyl moiety connects to protein amino acid residues in the intestinal cells of mosquito larvae, and thus the Mtx1 would disable protein function and kill the epithelial cells.

Cry4Ba

Cry protein 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 could assist toxin to penetrate into cell membrane, leading to the formation of ion-permeable pores and eventually causing osmotic lysis of target cells.

Why do we put them together?

If they appear at the same time, they can be functional for a longer time and be effective to more mosquito species, which is also called synergism.

Thus, we tried to express the two genes together in E.coli and conduct a toxin test to compare the toxicity of each protein and their combination.

How to complete this plan?

This is an overview of our project, first of all, we extracted genome of Bt.i and Bs, and then use pcr to amplify the target gene ( Cry4Ba and Mtx1), These target sequences were then assembled into the expression vector pEASY-Blunt and then transformed into E.coli BL21. 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 Cry4Ba and Mtx1 could be induced by IPTG, and both of them would be purified using nickel column due to the his-tag.

In order to detect the expression level of proteins, we have designed a Mtx1, Cry4Ba and eGFP fusion protein. Linker peptides with sequence of Tryptophan – Tryptophan – Tryptophan – Serine can connect the two proteins without influencing activity of the two proteins. The function of this fusion protein is: once the Cry4Ba express, the GFP will express together. So we can know whether our Cry4Ba had expressed or not and detect the expression level by measuring the Green fluorescence force.

So as to conduct the toxicity test, we designed this special device . We mixed Cry4Ba protein and Mtx1 protein with the crushed cat food, for mosquito larvae are fond to eat cat food according to our observation. Once mosquitoe larvae eat this bait, the toxin proteins will get inside to the stomach or intestine of larvae and kill them. The toxic cat food cannot be threw into the water directly, for they can form a layer of grease which may cause the death of aquatic organisms due to asphyxia, so after mixing toxic proteins and cat food together, we will embed them by agarose. The function of agarose is to keep the poison cat food in shape and hydrophobic .

To demonstrate the toxicity of our Engineered E.coli, we did two toxicity tests with concentration of Cry4Ba E.coli and kind of toxin as their variable respectively. Our test system include 50mL ddH₂O and 20 2^nd instar Aedes albopictus larvae . The bacterial was mixed with 0.2g cat food and embedded by 100uL agarose to form gel-like “bait”. Each test group duplicated for 3 time.

Toxicity Test

To demonstrate the toxicity of our Engineered E.coli, we did two toxicity tests. Our test system include 50mL ddH₂O and 20 2^nd instar Aedes albopictus larvae. The bacterial was mixed with 0.2 g cat food and embedded by 100uL agarose to form gel-like “bait”.

1. Variable: Concentration of Cry4Ba E.coli

Test the toxicity of five different concentrations(1%,2%,3%,4%,5%), each concentration has three repetitions. Calculate the death rate in eight time points(2h, 4h, 6h, 8h, 10h, 12h, 24h, 48h).

2. Variable: Kinds of toxin

Test the toxicity of three toxins (Bt.i, Bs, Cry4Ba E.coli, BL21 E.coli) and one control group (replace bacterial by water). Both toxins and water are equal to concentration of 5% in the whole system