Part:BBa_K2224002:Experience

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Applications of BBa_K2224002

iGEM2017_SMS_Shenzhen conducted two experiments on part BBa_K2224002.

First is enzyme activity essay. Second is to conduct a Scanning Electron Microscopy of test the enzyme on shell (containing high concentration of protein) of dead scale insect. However, due to our misconduct during preserving the enzyme activity essay kit, we failed in getting the accurate enzyme activity factors. Behind is the SEM result.

Scanning Electron Microscopy

Purpose

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.

Method

In this experiment, in order to find if our protease can decompose the protein layer of scale insect and how effective this Protease can be on the insects’ shells，we submerged scale insects (dead and we have resolved its wax layer)in our protease for 72h (37℃）and used Electron Microscopy to scan the surface of shells.

Here are specific steps of our experiment: 1：Preparation of scale insect samples. We caught 6 identical look scale insects from Morning glory. After being disinfected, the shell portions of the scale insect were then cut down, which will be used later in the experiment. And at this time, the scale insects were killed.

2: Prepare Protease. We cultured E. coli that contains Protease gene, then we used 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, we got Protease solution and fracted cells. Then centrifuged solution for 10 min(25℃，13000rpm)

(The Ultrasonication we used) (The solution after centrifuging, and Protease is the clear liquid)

3：Eliminate the Wax on scale insects’ shells. We used alcohol solution (C=75%) to soak scale insects for 1h and killed them in this step. [The wax can solute in alcohol] (After eliminating the wax on the shell, we got the scale insects that just covered with protein.)

4: Use culture two dishes to take scale insects (Each dish takes three scale insects). Then we dropped the Protease that we got in the first step into the first dish that we called it: No.1 until the scale insects were covered by liquid. For another dish that we called it: No.2, we dropped water into it until the liquid covered the scale insects.

5: Move these dishes to incubator that held temperature in 37℃. We waited for 72h and then took out the dishes.

6: The student of Shenzhen university helped us to scan the surface of scale insects by using Electron Microscope (S100).

Pictures and Analysis

Fig.1

Fig.1 - Experimental group

Fig.2

Fig.2 - Control group：without protease

The insect body that was soaked by protease was very different from that of the control group. In the control group, the insect 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.

More Details:

Fig.3

Fig.3 - Details of experiment group

Fig.4

Fig.4 - Details of control group

The insect body of the experimental group (arrow marker) has obvious erosion marks, compared to the control group, which is relatively complete and flat.

Conclusion:

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 (using living scale insects）, in which we are not allowed to carry out experiment at present due to the safety policy.

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