Part:BBa_K2788010

PgpdA-HsbA-TtrpC

This part consists of three basic parts that can be directly linked to the fungal expression vector which can express in our fungus.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 903
  Illegal NheI site found at 2993
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 137
  Illegal BamHI site found at 3059
  Illegal XhoI site found at 693
  Illegal XhoI site found at 1075
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2553
  Illegal NgoMIV site found at 3695
  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 3409
  Illegal SapI site found at 315
  

iGEM2018 SZU-China

The HsbA 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 composite part was inserted 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.(Fig.2)

Fig.2 0.8%Agarose Gel Electrophoresis of DNA extracted from the positive clones and its validated by PCR. 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.

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 was electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining.(Fig.3)

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).

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)