Part:BBa_K4632002:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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.

UNIQc80461f0eff81611-partinfo-00000000-QINU UNIQc80461f0eff81611-partinfo-00000001-QINU