Difference between revisions of "Team:SZU-China/Demonstrate"

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We designed an experiment to investigate the difference between lethal period and mortality of our modified transformant and wild type <i>M.anisopliae</i>.
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We wonder if the products made from recombinant Metarhizium anisopliae are convenient and useful. For this reason, we have taken a step forward to verify the function of the modified strain and determine the suitable production method.
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In group infected by wild type <i>M.anisopliae</i>, we used 10μl emulsifiable powder in dose of 10<sup>9</sup> conidia/mL to infects 17 cockroaches individually. And in group infected by modified transformant, we used the same among of powder to infect 10 cockroaches individually.
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According to Professor Wang Zhongkang's suggestion, we chose emulsifiable powder as the product dosage form and non-woven fabric as the carrier of the product. Both of the above have the following advantages:
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/6/69/T--SZU-China--Demo1.jpg" />
 
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The results showed that wild type <i>M.anisopliae</i> toke effect after two days, while transformant took effects less than one day. For mortality each day, transformant showed above two times significantly higher (P <0.05) mortality than wild type. Two groups reached stationary state after four days, above 20% mortality in wild type, while 50% in transformant. </p>
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1. Compared with dry spores and water-dispersing agents, the emulsifiable powder has stronger storage stability, UV resistance and thermal stability.
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2. Compared with spore spray, non-woven fabric use less material and small-scale use.
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In order to ensure that we can produce milk powder in large quantities, Professor Wang took us to visit the Chinese largest fungal pesticide factory. The production process is shown in the figure.
 
 
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/c/c4/T--SZU-China--Demo-table1.png" />
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/2/28/T--SZU-China--Demo3.png" />
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Table 1: Experimental data of cockroaches infection experiment. * Table shows the number
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of cockroaches alive.
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It can be used after the emulsifiable powder is produced and brushed on the non-woven fabric. But we found problems: the non-woven fabric is so light that it maybe fly away. And the emulsifiable powder exposure to light may die easier.
  
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/8/8a/T--SZU-China--Demo-graph1.png" />
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Figure 1: Mortality of cockroaches between wild type <i>M.anisopliae</i> and transformant.
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We consulted the relevant literature and found that the Metarhizium emulsifiable powder emulsion is easy to fail under the sunlight, so we designed a box that can hold emulsifiable powder, called “Green Ground”. In order to verify the validity of the box(final generation), we designed an experiment, we first applied the emulsifiable powder to two non-woven fabrics, one in the box, we named it “With Box”, the other in the environment which called “Without Box”, and put an equal amount of cockroaches. After three days, the number of dead cockroaches in the two groups was counted. Repeated three times, the average mortality rate of the two groups was calculated and compared, and the mortality rate of the With Box group was higher. (As the chart shows)
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To solve this problem, we designed the GreenGround series, which has 3 generations of
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products (please click on Applied Design for details). We finally chose the third generation
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and patented it. In order to test the effect of the box on the killing effect, we did a
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comparative experiment and the results are as follows.
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Table 2: Mortality of cockroaches using or without using box.
 
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/c/cf/T--SZU-China--Demo2.png" />
 
<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/c/cf/T--SZU-China--Demo2.png" />
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Figure 2: Mortality of cockroaches using or without using box.
 
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To ensure that we can produce spore powder in large quantities, we visited Professor Wang Zhongkang, who told us about the fermentation production and took us to visit the factory, which provided conditions for us to produce products in the future.
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This shows that our GreenGround can maintain the lethal effect of Metarhizium anisopliae, so it’s necessary to use.
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Fermentation production process as shown：
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/2/28/T--SZU-China--Demo3.png" />
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/4/43/T--SZU-China--application_R3.jpg" />
 
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We used a series of produced emulsifiable powder in combination with our designed boxes to carry out a series of simulation experiments. According to Professor Wang Zhongkang's suggestion, we chose non-woven fabric as the carrier of the emulsifiable powder, because the emulsifiable powder can be attached to the non-woven fabric in a large amount, which greatly increases the probability of infection.
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The results showed that wild-type M.anisopliae toke effect after two days, while transformant took effects less than one day. For mortality each day, transformant showed above two times significantly higher (P <0.05) mortality than wild-type. Two groups reached stationary state after four days, above 20% mortality in wild-type, while 50% in transformant.
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After producing the emulsion, we applied it to the non-woven cloth and placed the non-woven cloth in area A of the box. Then screw the lid down as shown in the pic through the cylinder to form crevices which are also adjustable. The cockroaches can climb through the slope B and get in contact with Metarhizium anisopliae which will lead to death.</p>
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We recommend changing a pack of emulsifiable powder every week.
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<div class="row">
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<div class="col-10 offset-1">
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<div class="text-center">
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<caption>
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Table 1: Experimental data of cockroaches infection experiment. * Table shows the number
 +
of cockroaches alive.
 +
</caption>
 +
</div>
 +
<div class="card">
 +
<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/c/c4/T--SZU-China--Demo-table1.png" />
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</div>
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</div>
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/6/6e/T--SZU-China--application_2.jpg" />
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/8/8a/T--SZU-China--Demo-graph1.png" />
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</div>
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<div class="text-center">
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<caption>
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Figure 1: Mortality of cockroaches between wild type <i>M.anisopliae</i> and transformant.
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</caption>
 
</div>
 
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</div>
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Revision as of 23:22, 17 October 2018

Demonstrate

We wonder if the products made from recombinant Metarhizium anisopliae are convenient and useful. For this reason, we have taken a step forward to verify the function of the modified strain and determine the suitable production method.

According to Professor Wang Zhongkang's suggestion, we chose emulsifiable powder as the product dosage form and non-woven fabric as the carrier of the product. Both of the above have the following advantages:

1. Compared with dry spores and water-dispersing agents, the emulsifiable powder has stronger storage stability, UV resistance and thermal stability.

2. Compared with spore spray, non-woven fabric use less material and small-scale use.

In order to ensure that we can produce milk powder in large quantities, Professor Wang took us to visit the Chinese largest fungal pesticide factory. The production process is shown in the figure.

It can be used after the emulsifiable powder is produced and brushed on the non-woven fabric. But we found problems: the non-woven fabric is so light that it maybe fly away. And the emulsifiable powder exposure to light may die easier.

To solve this problem, we designed the GreenGround series, which has 3 generations of products (please click on Applied Design for details). We finally chose the third generation and patented it. In order to test the effect of the box on the killing effect, we did a comparative experiment and the results are as follows.

With box Without box
Group 1 10 4
Group 2 9 2
Group 3 12 5
Mean 9.5 3.0
Variance 2.3 2.3
Mortality 34.44% 12.22%

This shows that our GreenGround can maintain the lethal effect of Metarhizium anisopliae, so it’s necessary to use.

The results showed that wild-type M.anisopliae toke effect after two days, while transformant took effects less than one day. For mortality each day, transformant showed above two times significantly higher (P <0.05) mortality than wild-type. Two groups reached stationary state after four days, above 20% mortality in wild-type, while 50% in transformant.

Table 1: Experimental data of cockroaches infection experiment. * Table shows the number of cockroaches alive.
Figure 1: Mortality of cockroaches between wild type M.anisopliae and transformant.