# Team:Tongji China/Demonstrate

Tongji iGEM - Demonstrate
Tongji iGEM
Tongji iGEM
Demonstrate
Here is what we have accomplished with this project
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#### The Project

Our project aims to control the population of Diptera insects, such as mosquitos, which do harm to human health. Among all the Diptera insects Drosophila melanogaster is the only model creature with well developed protocols to culture and experiment, so we choose it as the chassis to test our design in the project.

It would be great for us to get accurate changes of the whole population in the natural environment to test our system. But, unfortunately, it is almost impossible for us to observe a small group in a specific area due to the lack of equipment and skills. Although Drosophila melanogaster has been regarded as a model creature for a long time, we have found little published data about its population changes.

Therefore, building a population growth model can help us determine the effect of our system when applied to natural environments.

#### Model Theory & Experiments

In 1945, Leslie P H. introduced a mathematical method to predict the age structure and population through time from the age structure of the initial population. Here we use Leslie matrix to make our model better.

Depending on the physiological characteristics of each individual, the maximum life age is divided into M groups. The objective it to find how the age distribution evolves. The time starts from t=0 and evolves in steps (t=0, 1, 2...), and the interval is the same as that of the age group.

Suppose that, at the beginning (t=0), the number of individuals in the I age group was Ni(0), i= 1, 2,... m. so the age distribution vector is:

$$\overrightarrow n(0) = [n_1(0), n_2(0), …, n_m(0)]^T$$
The reproductive rate of the I age group is fi(0) ,i= 1, 2,... m;
The survival rate is Si(>0), i= 1, 2,... m - 1;
Between two periods, there is an iterative relationship between the number of individuals in each age group ni:

$$\begin{cases} n_1 (t + 1) = \sum_{i=1}^m f_i \cdot n_i(t) = f_i \cdot n_1 (t) + f_2 \cdot n_2(t) + … + f_m \cdot n_m (t) \\ n_i (t + 1) = S_{i-1} \cdot n_{i-1}(t) \qquad i = 2, 3, …,m \\ \end{cases}$$
In matrix form:

$$L=\begin{bmatrix}{} f_1 & f_2 & ... & f_{m-1} & f_m\\ S_1 & 0 & 0 & 0 & 0\\ 0 & S_2 & 0 & 0 & 0\\ 0 & 0 & ... & 0 & 0\\ 0 & 0 & 0 & S_{m-1} & 0 \end{bmatrix}$$
We detected the male-male courtship of modified flies and the modified flies’ gender preferences when raising the temperature.
The results are as follows:
Figure 2 - The mating index of the treated group rises significantly at 29°C.
[time=5minutes, n=5, P<0.01]
Figure 3 - The modified fruit flies had no preference for males and females when mating.
[time=10minutes, n=8, P>0.5]

From the results above we can see that the mating index (the relative time that the fruit fly use for mating) for wide-type male flies of treated group rise significantly in In 29°C [n=5, P<0.01] and the modified male flies had no preference for males and females when mating [n=8, P>0.5].

Combining experimental data with data from previous literature on fruit fly reproduction and survival, we established relevant models and predicted the control results of fly’s populations.
The following chart shows the differences between the proportions of female flies that can produce normal offspring in two different conditions:

It can be seen that in the case of the small mating rate, the inhibition effect of releasing sterile male flies with male-male courtship is more obvious to reducing population quantity, which is consistent with the purpose of our modeling.

Here we also shows the result when the number of modified fruit flies was five times as large as the wild flies and the mating rate is 0.4, the effect on the final population in 15 days. (The yellow line represents the sterile male flies with male-male courtship, and the purple line represents the sterile male flies)

More details

Based on the historical data and experimental observations, we simulated the population change of both modified and wild type Drosophila melanogaster in natural environment. It indicates that the introduction of modified Drosophila into wild populations can indeed control the population of the group even in one or two weeks. These results that it may be a viable and novel population control method.

Given the potential impact on the mating and viability of transgenic flies, we did a series of related experiments.
We found the reproductive curve of the modified fruit flies:

Figure 8 - The reproduction curve of the modified fruit flies
[n=5]

The “wild type” fruit flies have lived in laboratory environment with enough food and space for many generations, so its competitive power is lower than the fruit flies that live outdoors. In the experiment for testing the reproductive capacity, we found that control groups did not have offspring at 29°C. We thought that the mating ability of the “wild type” fruit fly lived in laboratory could be affected severely in 29°C while the modified flies would be less affected.

So we designed experiment 8, and the result supports our hypothesis.

Figure 9 - The impact that proportion of modified flies have on mating rates of population
[n=5]

After we transfer the gene to the fruit fly, they regain the capability of reproductive, and the modified fly is stronger than the control groups according to experiment 6.
The number of live flies in the absence of food and water:

Figure 5 - The survival curve of fruit flies in the absence of food and water

The number of live flies in high temperatures (37°C):
Figure 6 - The survival curve of fruit flies under high temperature
[T=37°C]

The seismo-tube experiment:
Figure 7 - The modified fruit flies crawl higher in the seismo-tube experiment

As a result, we suppose that when we transfer our system into flies live in natural environment, their survivability, mating capacity and competitive power may be higher than other wild-type flies, which may have a more favorable effect on our model.

#### Future

In the future we are planning to introduce new parts to strengthen the effect by introducing infertile male flies along with the existing male-male courtship features. We can also screen out more GAL80 temperature-dependent variants to make our system work at different temperatures. Furthermore, we are trying to combine the different parts in our system into only one chromosome to enhance the heritability. We sincerely hope that our work will eventually be useful in the real world.
Ignis Fly

Tongji_China iGEM 2017 Team
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