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 <i>Metarhizium anisopliae</i> are convenient and useful.
 +
 
 
</p>
 
</p>
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<p>
 +
Previously, emulsifiable powder as the product dosage form and non-woven fabric as the carrier suggested by Dr. Wang Zhongkang, can be proved that our product has stronger storage stability, UV resistance and thermal stability. Besides compared with spore spray, non-woven fabric use less material and small-scale use.
  
 +
</p>
 +
<p>
 +
For now, we have taken a step forward to verify our product has the functions including the ability of producing a great amount of spores, high mortality, and necessity of using GreenGround.
 +
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</p>
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<div id="Ability" class="indent">
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<h2>
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Ability of producing a great amount of spores:
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<p>
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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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In order to ensure that we can produce emulsifiable 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/2018/6/69/T--SZU-China--Demo1.jpg" />
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/2/28/T--SZU-China--Demo3.png" />
 
</div>
 
</div>
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<caption>
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Fig 1: The emulsifiable powder production flow chart
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</caption>
 
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</div>
 
</div>
 
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<div class="row">
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<p>
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It can be used after the emulsifiable powder is produced and brushed on the non-woven fabric.
  
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</p>
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</div>
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<div id="mortality" class="indent">
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<h2>High mortality</h2>
 +
<p>
 +
We designed an experiment to investigate the difference between lethal period and mortality of our modified transformant and wild-type <i>M.anisopliae</i>. (According to the non-release principle, the experiment only tested the virulence of modified transformant and wild-type <i>M.anisopliae</i> in the laboratory.)
 +
</p>
 +
<p>
 +
In group infected by wild-type <i>M.anisopliae</i>, we used 10μl emulsifiable powder in a dose of 10^9 conidia/mL to infects 10 cockroaches individually. And in group infected by modified transformant, we used the same among of powder to infect 10 cockroaches individually.
 +
</p>
 
<p>
 
<p>
−
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>
+
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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<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.
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</caption>
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<div class="card">
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/1/14/T--SZU-China--Demo-0-1.png" />
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Table 1: Experimental data of cockroaches infection experiment. * Table shows the number
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Fig 2: Mortality of cockroaches between wild type <i>M.anisopliae</i> and transformant.
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of cockroaches alive.
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<img class="card-img-top" src="https://static.igem.org/mediawiki/2018/1/1c/T--SZU-China--cockroach.jpg" />
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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>
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</div>
 
</div>
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<caption>
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Fig 3：The cockroach which covered by spores
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</caption>
 
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<div id="Necessity" class="indent">
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<h2>Necessity of using GreenGround</h2>
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<p>
 
<p>
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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)
+
We found some problems that the non-woven fabric is so light that it maybe fly away, and the emulsifiable powder exposure to light may die easier.
  
 
</p>
 
</p>
 +
<p>
 +
To solve this problem, we designed the GreenGround series, which has 3 generations of products (please click on <a style="color: #469789;" href="https://2018.igem.org/Team:SZU-China/Applied_Design">Applied Design</a> 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.
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Figure 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" />
 
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Fig 4: Mortality with or without 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.
+
This shows that our GreenGround can maintain the lethal effect of <i>Metarhizium anisopliae</i>, so it’s necessary to use.
  
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</p>
 
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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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Fig 5：The third generation of GreenGround
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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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</p>
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<p>
 
<p>
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<p>
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Because of the Do Not Release Policy, we couldn’t test our transformant in our GreenGround. But from the evidence above we can see that, it has the ability of producing a great amount of spores, high mortality, and necessity of using GreenGround. We believe that if we combine our spores and GreenGround together, the product would work well.
−
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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Reference：
 +
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<p>
 
<p>
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We recommend changing a pack of emulsifiable powder every week.
+
[1]Yin Youping, Wang Zhongkang, Xie Ning, et al. 2009. Insecticidal fungi emulsifiable powder:, CN101564047[P].
 
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<a class="nav-link" href="#Ability">Ability of producing spores</a>
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<a class="nav-link" href="#mortality">High mortality</a>
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Latest revision as of 03:08, 18 October 2018

Demonstrate

We wonder if the products made from recombinant Metarhizium anisopliae are convenient and useful.

Previously, emulsifiable powder as the product dosage form and non-woven fabric as the carrier suggested by Dr. Wang Zhongkang, can be proved that our product has stronger storage stability, UV resistance and thermal stability. Besides compared with spore spray, non-woven fabric use less material and small-scale use.

For now, we have taken a step forward to verify our product has the functions including the ability of producing a great amount of spores, high mortality, and necessity of using GreenGround.

Ability of producing a great amount of spores:

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

Fig 1: The emulsifiable powder production flow chart

It can be used after the emulsifiable powder is produced and brushed on the non-woven fabric.

High mortality

We designed an experiment to investigate the difference between lethal period and mortality of our modified transformant and wild-type M.anisopliae. (According to the non-release principle, the experiment only tested the virulence of modified transformant and wild-type M.anisopliae in the laboratory.)

In group infected by wild-type M.anisopliae, we used 10μl emulsifiable powder in a dose of 10^9 conidia/mL to infects 10 cockroaches individually. And in group infected by modified transformant, we used the same among of powder to infect 10 cockroaches individually.

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.
Fig 2: Mortality of cockroaches between wild type M.anisopliae and transformant.
Fig 3：The cockroach which covered by spores

Necessity of using GreenGround

We found some problems that 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%
Fig 4: Mortality with or without box

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

Fig 5：The third generation of GreenGround

Because of the Do Not Release Policy, we couldn’t test our transformant in our GreenGround. But from the evidence above we can see that, it has the ability of producing a great amount of spores, high mortality, and necessity of using GreenGround. We believe that if we combine our spores and GreenGround together, the product would work well.

Reference：

[1]Yin Youping, Wang Zhongkang, Xie Ning, et al. 2009. Insecticidal fungi emulsifiable powder:, CN101564047[P].