Regarding contributions,we made two biological parts，firstly，in prokaryotes, RNase III is the main degrading enzyme of dsRNA. In order to increase the accumulation of dsRNA expression in Pseudomonas chlororaphis,we knocked out RNase III gene of Pseudomonas chlororaphis and obtained mutant P.chlororaphis KC-P can greatly increase the accumulation of dsRNA.
Secondly，in order to ensure the biosafety of our engineered bacteria, we introduced LacI operons into plasmids that can express the Plagiodera versicolora actin protein dsRNA to regulate the expression of dsRNA. In addition, on the basis of the introduction of LacI operons, we successfully integrated the KillerRed protein expression frame to construct the KC5 plasmid, which further ensured the safety of our construction of engineered bacteria.
Besides,we independently designed and made the insect gripping device of Plagiodera versicolora. After continuous iteration and update, we finally made the insect gripping device of Plagiodera versicolora combined with practical application.Secondly, in terms of modeling, we used the Leslie matrix model to simulate the population dynamics and quantity forecast of Plagiodera versicolora, discussed its quantity changes, and predicted its growth trend.We hope that the content of our project will help future iGEM teams improve the efficiency of their construction experiments, providing some practical lessons for other iGEM teams.
The parts we built are shown in the following table,You can click on the part number to see the details.
| Part Numeber | Name | Type | Part Description |
|---|---|---|---|
| BBa_K4832003 | P.chlororaphis KC-P | Basic | A P.chlororaphis that knocks out the RNase III gene can greatly improve the accumulation of dsRNA |
| BBa_K4832004 | KC5 | Basic | A plasmid containing LacI operon and KillerRed kill switch to express Plagiodera versicolora actin protein dsRNA |
Insect gripping device of Plagiodera versicolora
There is no standard insect gripping device on the market, and homemade brush is generally used as a insect gripping device in the laboratory, but homemade brush as a insect gripping device has different advantages and disadvantages for different growth periods of Plagiodera versicolora. The first instar larvae are short in body length, which is easy to hurt the larvae when using the ordinary brush. The second and third instar larvae are moderate in size, and the damage is less when using the brush, and the Plagiodera versicolora larvae will not be pressed to a greater extent when taking the insects.
In order to facilitate the selection of insects during the bioassay process, we have optimized the first-generation of stick-shaped insect brush.Inspired by the disposable dropper in the laboratory, we replaced the stick that fixed the bristles with the dropper, glued the bristles to the different sizes of the pipette head without changing the part of the dropper device, and then assembled it, so as to achieve the expected idea, and made a second-generation insect brush with suction capsule.In order to make the larvae in the dark environment easy to be found, and then made the third-generation illuminated insect brush.We use Light-Emitting Diodes（LED），and then changed the upper end of the third-generation dropper into a push-action pen tube, assembled the button battery and LED in the pen tube to make a small flashlight, and added bristles at the front end.
Figure 1A: illuminated insect gripping device
Figure 1B: picture of real product
After a series of the mentioned schemes’ design and verification, our core device design is finally determined. Under the investigation of the actual situation, the second-generation and third-generation insect brush are retained for the different tasks of insect gripping, which are already in use. Constant iterations have resulted in a convenient, lightweight, low-cost and reusable insect gripping device. The production of this insect brushes is only a small step forward for our bioassay process, but when our design is accepted by other research teams that need insect gripping, it will represent a big step for us. Therefore, our research on insect gripping devices will not stop at this bioassay, but continue to be people-oriented and polish products, in order to let really useful insect gripping devices walk up to the world.
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In our work, we tested the effectiveness of our product using the Plagiodera versicolora .In order to better predict and guide the experiment, we establish a mathematical model of the population dynamics of the Plagiodera versicolora with an age structure, discuss its population changes, and predict its growth trend.
Due to the fact that population growth is not only related to the total population size, but also to the age structure of the population, we have established a discrete model that considers the age structure of the population, the Leslie matrix model. Based on existing experimental data, the Leslie model was used to simulate the population dynamics and quantity prediction of the Plagiodera versicolora, and compared with measured data.Read about it
Figure 2 : Population age distribution of Plagiodera versicolora
Our model fits well with the data collected in the laboratory, and it can be seen that the survival rate of the Plagiodera versicolora is between 60% -70% under laboratory conditions. In future experiments, we will introduce a new variable based on this, which is to use our product. Comparing the results before and after introducing this factor can reflect the effectiveness of our product to a certain extent.
All of this may be helpful to other iGEM teams. We hope it will do something for the iGEM community.