This year, in our project, we constructed a new expression vector working effectively in the fungus and submitted our parts and transforred into Metarhizium anisopliae 128 to increase the adhesion, penetration and immune avoidance capacity of the modified Metarhizium anisopliae. Therefore, we performed experiment on three aspects and the experimental characterizations of our parts are shown as follow.

Adhesion：PgpdA-HsbA-TtrpC

In this part, PgpdA is a strong promoter that allows the HsbA protein to be expressed without induction.

The HsbA( BBa_K2788000 ) from Beauveria bassiana encodes a kind of membrane surface hydrophobic protein which helps our spores adhere to the wax on the cockroach body surface. Moreover, with the overexpression of HsbA, our spores can more effectively adhere to the cockroach. Then it will follow as spores’ germination, germinal tube, appressorium and the next penetrating process.

This part was insert into the expression vector by restriction sites EcoRI and PstI (Fig.1), and the correct construction of this recombinant plasmid was confirmed by PCR identification and sequencing of the PCR products.

Fig.1 Construction of expression vector HsbA-pBC. PgpdA and TtrpC come from parts of 2016_NYMU-Taipei: BBa_K2040101 and BBa_K2040102 , and HsbA comes from the Beauveria bassiana ARSEF 2860. The PgpdA-HsbA-TtrpC part is connected to the pBC plasmid through the BioBrick site.

We transformed the expression vectors into Metarhizium anisopliae 128 by the method of Xiaoling Wang, and the positive clone was confirmed by G418 sulfate screening and nucleic acid electrophoresis.

The transformed strain Metarhizium anisopliae 128 was grown in 1/4 SDAY liquid medium, and obtain total protein by FastPrep and ultrasonic crushing. The lysate was then centrifuged and the supernate were electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining.(Fig.3)

Besides, by using the method of compared four areas of each wing in the scanning electron microscope before and after treatment which is ‘put the petri dishes on WD-9405B horizontal shaking table and opened the lowest rolling speed for 10min (to make the spores on the wings evenly impacted by the water flow)’ in the HsbA macro verification protocol, we can finally compared whether there was any change in the position and number of spores in the observing area. (illustrated with Fig.4 and Fig.5)

Fig.4 One of the wild-type Metarhizium anisopliae 128 groups: P1 is the picture before treated and P2 is the picture after treated. The spores quantity is 40 in P1 and 102 in P2. The change rate of spores’ quantity is (102-40)/102=60.8%

It’s obvious that it happens great change in the position and number of spores in the observing area of wild-type Metarhizium anisopliae 128 experimental groups.

Fig.5 One of the Metarhizium anisopliae HsbA transformant groups: P3 is the picture before treated and P4 is the picture after treated. The spores quantity is 42 in P3 and 52 in P4. The change rate of spores’ quantity is (52-42)/52=19.2%

It’s also obvious that it happens no much change except the place circled in red in the position and number of spores in the observing area of Metarhizium anisopliae HsbA transformant experimental groups.

Therefore, this result well confirmed that Metarhizium anisopliae HsbA transformant certainly enhanced the capacity of adhesion.

Finally the following chart(Fig.6) can be obtained by statistical data of four areas in all experimental groups.

Fig.6 Ma128 is the wild-type Metarhizium anisopliae 128 groups, and the change rate of spores’ quantity is 41.0%. HsbA is the Metarhizium anisopliae 128 HsbA transformant groups, and the change rate of spores’ quantity is 9.3%.

Penetration: Bbchit

In order to make Metarhizium anisopliae penetrate the corpus callosum more efficiently, we transferred Bbchit( BBa_K2788001 ), a chitinase gene from Beauveria bassiana ARSEF 2860, which encodes a chitinase that can be secreted extracellularly. The chitin of the body surface decomposes, thereby destroying the wall of the corpus callosum, so that the genus Metarhizium enters the sputum.(Fig.7)

Fig.7 Construction of expression vector Bbchit-pBC. PgpdA and TtrpC come from parts of 2016_NYMU-Taipei: BBa_K2040101 and BBa_K2040102, and Bbchit comes from the Beauveria bassiana ARSEF 2860. The PgpdA-Bbchit-TtrpC part is connected to the pBC plasmid through the BioBrick site.

We have a part encoding Chitinase, the Bbchit express and function intracellularly. We constructed a shuttle vector to transform this part and the positive clone was confirmed by G418 sulfate screening and nucleic acid electrophoresis.(Fig.8)

The crude enzyme solution was obtained by cell disruption using ultrasonic, followed by SDS-PAGE protein electrophoresis and Coomassie blue staining.(Fig.9)

To determine the activity of chitinase, we improved it according to the DNS colorimetric method of Kan Zhuo, Xiaozhen Shi. First, the standard curve was drawn with different concentration gradients of glucose solution, and 0.5 ml of the wild-type and transformed type 1, 3, 5, 7, and 9 days of culture solution were respectively taken for enzyme activity test: the crude enzyme obtained after filtering the culture solution was used. The solution was mixed with 0.5 ml of 1% chitin colloid, reacted at 37 ° C for 60 min, and then added to a 0.5 ml DNS boiling water bath for 10 min. The absorbance of the obtained product was measured and the enzyme activity was calculated. There were three groups of wild-type and transformed type. Parallel, three parallel experiments were performed in each group, and the final data were averaged.

We calculate activity based on the standard curve formula: U=（A540+0.03279）/2.202 (Fig.10), a summary of the data at different times is made into a line chart as follows.We can see that after 9 days the transformed type’s enzyme activity is still growing and the wild-type is falling.(Fig. 11)

Fig.10 Glucose standard curve

Fig.11 changes in chitinase activity over time.

In order to verify the function of Bbchit from a macro level, we improved Kan Zhuo's chitin transparent circle method for verification. We stained the czapek solid medium without chitin colloids in red with 0.1% Congo red dye solution and then cultured wild-type and transformed Metarhizium. Compared with the size of the colony, the size of the transparent circle was compared to obtain the transformed type. The size of the transparent circle is the diameter of the chitin transparent ring (R2) and colonies. The ratio of the diameter (R1), expressed as R2/R1.The conclusion that the chitinase activity of Metarhizium anisopliae is enhanced.

Fig.12 A: wild-type Metarhizium anisopliae 128 produced transparent zone on Czapek chitin - induced medium; R1: 8mm, R2: 9mm; R2/R1=9/8 B: Metarhizium anisopliae HsbA transformant produced transparent zone on Czapek chitin - induced medium; R1’: 8mm, R2’: 12mm; R2’/R1’=12/8=3/2

Therefore, these results well confirmed that the chitinase activity of Metarhizium anisopliae Bbchit transformant is about 1.3 times that of wild-type Metarhizium anisopliae 128. Our modified fungus certainly enhanced the capacity of penetration.

Immune avoidance: MCL1