Part:BBa_K4632002:Experience

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Experimental Results

1. Verifying the Expression and Secretion Proficiency of Cry3A-like Toxin

The OmpA-Cry3A-like toxin fragment on the plasmid (ordered from Guangzhou IGE Biotechnology Co.,Ltd.) was amplified using PCR and cloned into the pET-30a plasmid using the Gibson Assembly method (2× MultiF Seamless Assembly Mix kit, ABclonal) to obtain the plasmid pET-30a-OmpA-Cry3A-like toxin. Subsequently, this plasmid was transformed into E. coli BL21(DE3) to test the secretion expression of the toxin. The pET30a-OmpA-Cry3A-like toxin was cultured overnight in LB broth, and the culture was induced with IPTG for 3 hours. The culture was then centrifuged at 6,000 rpm for 10 mins to separate the bacterial cells and the supernatant, and the expression results were analyzed by SDS-PAGE.

Figure 3 SDS-PAGE Electrophoresis Detection of Cry3A-like Toxin Expression. Lane 1: Concentrated protein supernatant of pET-30a (+IPTG); Lane 2: Concentrated protein supernatant of pET-30a-OmpA-Cry3A-like toxin (+IPTG); Lane 3: Concentrated protein supernatant of pET-30a-OmpA-Cry3A-like toxin (-IPTG)

The supernatant of the induced culture showed a 66.6 kDa band corresponding to Cry3A-like toxin, which was absent in the supernatant without IPTG induction and the wild-type control (Figure 3). This indicates the successful secretion expression of Cry3A-like toxin.

Next, a 6×His tag will be added to pET30a-OmpA-Cry3A-like toxin using the enzyme cutting connection/ΩPCR method and Western blot will be performed to further confirm the secretion expression of Cry3A-like toxin.

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