Difference between revisions of "Part:BBa K2788000"

Revision as of 07:09, 11 October 2018

HsbA_Beauveria bassiana ARSEF 2860.

Hydrophobic Surface Binding Protein A is called for short HsbA. It forms a protective layer on the surface of the bacteria to form an aerial structure, reducing the surface hydrophobic pressure and promoting the interaction between the fungus and the host. In our project, HsbA protein helps our fungus stick more firmly to the body surface of cockroach and makes infection more effectively.

Sequence and Features

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 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 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.

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).