Part:BBa_K2788011

PgpdA-Bbchit-TtrpC

This part consists of three basic parts that can be directly linked to the fungal expression vector.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 903
  Illegal NotI site found at 2890
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 137
  Illegal BamHI site found at 3365
  Illegal XhoI site found at 693
  Illegal XhoI site found at 1075
  Illegal XhoI site found at 2877
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2559
  Illegal NgoMIV site found at 3088
  Illegal NgoMIV site found at 4001
  Illegal AgeI site found at 1282
  Illegal AgeI site found at 1430
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1109
  Illegal BsaI.rc site found at 3715
  Illegal SapI site found at 315
  

iGEM2018 SZU-China

In order to make Metarhizium anisopliae penetrate the corpus callosum more efficiently, we constructed an expression vector containing part Bbchit(Fig.1)

Fig.1 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 constructed a shuttle vector to transform this part and the positive clone was confirmed by G418 sulfate screening and nucleic acid electrophoresis. (Fig.2)

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 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.

The crude enzyme solution was obtained by cell disruption using ultrasonic, followed by SDS-PAGE protein electrophoresis and Coomassie blue staining. (Fig.3)

Fig.8 SDS-PAGE analysis of membrane protein of wild-type Metarhizium anisopliae 128 and modified Metarhizium anisopliae 128. Lane M: Marker Ladder；Lane 1：Metarhizium anisopliae 128；Lane 2: recombinant strain Metarhizium anisopliae 128. Lane 2 showed the band(in the red box) corresponded with the molecular weight of Bbchit(38kDa).

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, 9 and 12 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℃ 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.4), 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.5)

Fig.4 Glucose standard curve.

Fig.5 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.6)

Fig.11 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.