Electron Microscope Observation of Erosion of Scale Insect’s Shell after Introducing Protease
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.
[Safety Highlight]: We absolutely understand the importance of safety in this experiment since it involves insect use, and we are strictly following safety principle in this experiment. After discussing with the iGEM Safety Committee, we confirmed that we could only shells from scale insects instead of real alive scale insects to avoid potential issues. Therefore, in this experiment, only scale insects' shells are used.
By doing this experiment, we hope to find how effective this protease is so that we can know how effective our project will be. Besides, electron microscopy can provide a detailed description of the result of protease erosion, and by comparing the scanning images, we would have a better understanding of how our project would function on real scale insects.
III Design & Procedure
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.
Result Picture 1 - Experimental Group
Result Picture 2 - Control Group (without protease)
The shell soaked in protease appears to be very different from that in the control group.
In control group, structure was relatively complete and the characteristics were clear, but the insect body of the experimental group had lost its structural characteristics under the action of protease, and the outer shell was severely eroded.
Result Picture 3 - Experimental Group
Result Picture 4 - Control Group (without protease)
The sample in the experimental group (arrow marker) has obvious erosion marks compared to the control group, which is relatively complete and flat.
V Conclusion & Implication
Basing on our experiment result that experimental group shows significant erosion, we prove that our protease can degrade scale insect’s protein layer on its shell under ideal conditions. Thus, we can conclude that our Proteinase is expressed successfully in the E. coli expression system, and has the ability to degrade the protein structures on scale insect. This experiment sets stage for our future experiment to test whether this enzyme is able to work under real condition, in which we are not allowed to carry out experiment at present due to the safety policy.