Electron Microscope Observation of Erosion of Scale Insects’ Shells after Introducing Protease

Background:

The purpose of our project is to erode the insects’ shells so that these pests can be eliminated by the pesticides that have a lower toxicity. Our Protease’s gene came from a kind of Fungi named Verticillium lecanii. We have transferred this gene into E. coli, and we have found that this Protease that produced by E. coli was active.

Purpose of This Experiment:

We want to find how effective this Protease can be on the insects’ shells so that we can adjust our final product in the future.

Design of This Experiment:

1. Catch some scale insects. We prepared 6 scale insects that used in this experiment and we got them from a morning glory plant.

2. Prepare protease: culture E. coli that contains Protease gene, then use Ultrasonication and centrifuge to take out Protease.

1.7ml -50mM-ph8.0-Tris-HCl Solution + Precipitation (17ml E. coli broth after centrifuging triple times).

After using Ultrasonication, protease solution and fracted cells are got. Then centrifuge solution for 10 min (25℃, 13000rpm).

(The Ultrasonication we used)

(The centrifuged solution, and protease is the clear liquid)

3. Eliminate the wax on scale insects’ shells. Use alcohol solution (C=75%) to soak scale insects for 1 hour and killed them, since the wax can also solute in alcohol. After eliminating the wax on the shell, scale insects that just covered with protein are got.

4. Use two culture dishes to take scale insects, and each dish contained three insects. Then we dropped the protease we got from the first step into the first dish (No.1 Dish) until the scale insects were covered by liquid. For another dish (No.2 Dish), we dropped water into it until the liquid covered the scale insects.

5. Move these dishes to incubator of 37℃. Then waited for 72h and then took out the dishes.

6. Use Electron Microscope (S100) to get the result.