Fig.1 Construction of expression vector HsbA-pBC. PgpdA and TtrpC come from parts of 2016_NYMU-Taipei: <a href="http://parts.igem.org/Part:BBa_K2040101">BBa_K2040101</a> and <a href="http://parts.igem.org/Part:BBa_K2040102">BBa_K2040102</a> , and HsbA comes from the <i>Beauveria bassiana</i> ARSEF 2860. The PgpdA-HsbA-TtrpC part is connected to the pBC plasmid through the BioBrick site.

Fig.2 0.8%Agarose Gel Electrophoresis of DNA extracted from the positive clones and its identification by restriction digestion. The product of plasmid digested showed two signal bands at 335 bp and 741bp respectively, which correspond to the length of M.a primer PCR product and HsbA primer PCR product. Lane 1: M.a primer PCR product; Lane 2: HsbA primer PCR product; Lane M: DL marker.

Fig.3 SDS-PAGE analysis of membrane protein of wild-type Metarhizium anisopliae 128 and modified Metarhizium anisopliae 128. Lane M: Marker Ladder；Lane 128：Metarhizium anisopliae 128；Lane HsbA1 and HsbA2: recombinant strain Metarhizium anisopliae 128. Lane HsbA1 and HsbA2 showed the same band(in red box) corresponded with the molecular weight of HsbA(24kDa).

Fig.4 wild-type Metarhizium anisopliae 128: P1 is the picture before being rinsed and P2 is the picture after being rinsed. The spores amount is about 105 in P1 and spores amount is about 45 in P2. The retention rate is 45/105=42.86%

Fig.5 Metarhizium anisopliae HsbA transformant: P3 is the picture before being rinsed and P4 is the picture after being rinsed. The spores amount is 32 in P3 and spores amount is about 29 in P4. The retention rate is 29/32=90.63%

Fig.6 Construction of expression vector Bbchit-pBC. PgpdA and TtrpC come from parts of 2016_NYMU-Taipei: <a href="http://parts.igem.org/Part:BBa_K2040101">BBa_K2040101</a> and <a href="http://parts.igem.org/Part:BBa_K2040102">BBa_K2040102</a>, and Bbchit comes from the Beauveria bassiana ARSEF 2860. The PgpdA-Bbchit-TtrpC part is connected to the pBC plasmid through the BioBrick site.

Fig.7 0.8%Agarose Gel Electrophoresis of DNA extracted from the positive clones and its identification by restriction digestion. The product of plasmid digested showed two signal bands at 335bp and 1044bp respectively, which correspond to the length of M.a primer PCR product and Bbchit primer PCR product. Lane 1: M.a primer PCR product; Lane 2: Bbchit primer PCR product; Lane M: DL marker. </p>

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

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，a summary of the data at different times is made into a line chart as follows.

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.

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.

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