Team:SZU-China/Procedure

Procedure

Construction of plasmid vector

We used the fungal plasmid pBC (donated from professor Xie) for our project, which can propagate in Metarhizium anisopliae 128. The vector contains the following parts:

Gene Description
PgpdA PgpdA is the constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans.
TtrpC TtrpC is a tryptophan terminator from Aspergillus nidulans.
HsbA Encoded by the gene HsbA from Beauveria bassiana ARSEF 2860.
Bbchit Encoded by the gene Bbchit from Beauveria bassiana ARSEF 2860.
MCL1 MCL1_Metarhizium robertsii ARSEF 23
Tryptophan-MazF Suicide switch, induces apoptosis in the absence of tryptophan.

Inside the vectors，there are four gene pathways. PgpdA promoter starts the transcription of HsbA, Bbchit, MCL1, Ttyptophan-MazF. And TtrpC terminator terminates the transcription. It is well to be reminded that the pathway, PgpdA-Ttyptophan-MazF-TtrpC has a special switch, which induces apoptosis in the absence of tryptophan.

The model organism used in the project is Metarhizium anisopliae 128 (presented by professor Zhongkang Wang from Chongqing University), which is a major strain of Metarhizium anisopliae 128 with strong virulence.

Transformation and expression

We used the transformation method of Xiaoling Wang to transform Metarhizium anisopliae 128, and obtained stable transformant through the screening of G418 sulfate, and then verified the successful transformation through extracting genome, primer PCR and nucleic acid electrophoresis.

HsbA

To verify the existence and function of HsbA protein, the following treatment is done.

The transformed strain Metarhizium anisopliae 128 was grown in 1/4 SDAY liquid medium, and obtain total protein by smashing cells with FastPrep and ultrasonic crushing. And then we detected the native molecular mass by SDS-PAGE and coomassie blue staining. Besides, we spread the spores of Metarhizium anisopliae 128 and Metarhizium anisopliae HsbA transformant onto the cockroaches’ legs. After 16h germination, we took out and placed the cockroaches’ legs on the scanning electron microscope for observation. Then we rinsed the observed area on the cockroaches’ legs with 200ul ddH2O and observed them on the microscope again. Finally, we can compare whether is there any change in the position and number of spores in the observing area.

Bbchit

In order to verify the function of the transferred chitinase, the following experiments were performed.

The wild-type Metarhizium anisopliae 128 and Bbchit transformant were cultured in 1/4 SDAY liquid medium, and obtained the crude enzyme solution by smashing cells with FastPrep and ultrasonic crushing. Then, the native molecular mass of protein was detected by SDS-PAGE and Coomassie staining. In addition, to verify the enzyme activity, we used Czapek culture medium to culture the wild type and the modified type and took bacteria medium of 1, 3, 5, 7, 9 and 12 days to filter and obtain the crude enzyme solution. We optimized the method of Kan Zhuo‘s and Xiaozhen Shi’s to chart the chitinase activity curve through DNS colorimetry. At the macro level, using the method of transparent circle, we verified that our transferred chitinase can strongly penetrate the chitin of body surface of cockroach.

MCL1

In order to verify the ability of immune-avoidance in MCL1-transformant, the following experiments were performed.

The wild-type Metarhizium anisopliae 128 and MCL1 transformant were cultured in 1/4 SDAY broth. The expression of MCL1 was detected by quantitive PCR at the mRNA level and SDS-PAGE at the protein level. Besides, to compare the differences of immune-avoidance ability between wild-type and transformant M.anisopliae, we will inject hyphae homogenate into a cockroach and extract hemolymph in different time points. Then count the nodules under the light, nodules are formed by hemocytes surrounding pathogens. The number of nodules grows in proportion to the degree of immune response. Meanwhile, observe hemolymph smear under a phase contrast microscope. We confirmed that MCL1 can promote immune-avoidance.

Suicide Switch

To verify the function of Suicide Switch, and get the limited concentration of Tryptophan, the following experiments were done:

The transformed strain Metarhizium anisopliae 128 was grown in solid and liquid czapek with different concentrations of L-Tryptophan. After 6 days of cultivation, we compared the Metarhizium anisopliae in 10 different solid medium. We also detected the dry weight of Metarhizium anisopliae cultivated in 10 different concentration of 100mL liquid medium. Finally we can see the result that the transformation succeeded for a solid medium without Tryptophan could not survive. And we found the limited L-Trp concentration of the transformed strain Metarhizium anisopliae was 0.9%.

Application and realization

In order to make our Metarhizium anisopliae spores into products, we designed GreenGround, a containing box for spores, with a structure similar to the cockroach house.

Compared to regular chemicals, GreenGround reduces the elimination of cockroaches. To prove its usefulness, we built a simulated room to test it. But according to the principle of no release, we could not use the modified Metarhizium anisopliae. So in the experiment we used the wild type Metarhizium anisopliae.

We built two mini-houses using KT Board (made of polystyrene) to simulate two rooms, which connected with a corridor. We design control group without any drugs, and experimental group with cockroach killing chalk, BAYER-Premise and out product equipped with M.anisopliae emulsifiable powder. Then, we put thirty cockroaches into one room each time, and came back to see the results after three days. We focused more attention on migration rate and mortality.

Finally, we got results that the migration rate of control group was 7.78%, while 21.11%, 20.00%, 6.67% respectively in groups using cockroach killing chalk, BAYER-Premise and our product. For mortality in three days, 77.78% in group using chalk, 84.44% in group using Premise, 28.89% in group using our product, and 2.01% in group using nothing. Statistical Analysis in our model shows our product with M.anisopliae emulsifiable powder will not cause a high migration rate of cockroaches comparing with traditional products, although it has lower mortality within three days.

What’s more, we tested the mortality between groups using and without using our box to equipped M.anisopliae emulsifiable powder. The mortality in group using our box was 34.14%, while 12.22% in group without using any box. The result show that that it is necessary for us to use our Green-Ground box to hold M.anisopliae emulsifiable powder.

Migration Rate Mortality Gnawing Rate
Cockroach killing chalk 21.11% 77.78% 2.22%
BAYER-Premise 20.00% 84.44% 1.11%
M.anisopliae 6.67% 28.89% 11.11%
Control group 7.78% 1.11% 1.11%r