Part:BBa_K4632002
Cry3A-like toxin
Bacillus thuringiensis UTD-001, as well as the protoxin and toxin of this isolate, could be used to control pests such as fire ants, carpenter ants, Argentine ants, and Pharaoh's ants, including Solenopsis invicta(S. Invicta). Cry3A-like toxin isolated fromUTD-001 have been shown to be toxic to S.Invicta.(Bulla and Candas, 2003)
A. How does it work? It has been shown that after treatment with papain in vitro, the Cry3A-like toxin prototoxin (72.9 KD) forms an active toxin (66.6 KD) that is toxic to S. Invicta.(Bulla and Canda, 2003)
B. Bt toxin Cry3A-like protein: Eco-friendly and Safe Bt (Bacillus thuringiensis) is widely recognized as a safe and environmentally benign insecticide. And the Bt toxin Cry3A-like protein we used is Eco-friendly and Safe.(see more detail on https://2023.igem.wiki/scau-china/safety）
C.What we have done?（SCAU-China 2023）
In our design, we aimed to introduce a gene fragment encoding an active Cry3A-like toxin (66.6 kDa) into Escherichia coli using the pET30a vector to confer it with the ability to produce an active Cry3A-like toxin.
To achieve secretion expression, we added a signal peptide sequence, OmpA, to the N-terminus of Cry3A-like toxin. This was done to direct the transport of Cry3A-like toxin to the extracellular space. OmpA is a well-established signal peptide in Escherichia coli for the secretion expression of foreign proteins. Our SDS-PAGE results confirmed the successful secretion expression of Cry3A-like toxin.
Furthermore, we fused a 6×His tag to the C-terminus of Cry3A-like toxin to facilitate subsequent protein purification and Western blot-specific characterization experiments.
In addition, to validate the toxicity of the designed Cry3A-like toxin against red imported fire ants, we selected homologous receptors of Cry3A-like toxin known from NCBI in red imported fire ants. We then conducted molecular docking studies to assess the protein-protein interaction capability of Cry3A-like toxin, thus evaluating its toxic effects. Detailed results are presented in the characterization section.
Laboratory Characterization: To validate the expression and secretion capability of Cry3A-like toxin, we performed the following steps in the laboratory:
PCR Amplification: We initiated the process by amplifying the OmpA - Cry3A-like toxin fragment from the plasmid obtained from GUANGZHOU IGE BIOTECHNOLOGY LTD using PCR.
Cloning with Gibson Assembly: Subsequently, we cloned the PCR-amplified OmpA - Cry3A-like toxin fragment into the pET30(a) plasmid using the Gibson Assembly method with the ABclonal 2× MultiF Seamless Assembly Mix kit. This resulted in the creation of the plasmid pET30a-OmpA-Cry3A-like toxin (as depicted in plasmid map, Figure 1).
Transformation into E. coli BL21(DE3): Finally, we transformed the constructed plasmid pET30a-OmpA-Cry3A-like toxin into Escherichia coli BL21(DE3) to evaluate the secretion expression of the toxin protein.
These steps were taken to assess the ability of Cry3A-like toxin to be expressed and secreted in E. coli BL21(DE3).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1813
Illegal PstI site found at 295
Illegal PstI site found at 304
Illegal PstI site found at 1456 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1813
Illegal PstI site found at 295
Illegal PstI site found at 304
Illegal PstI site found at 1456 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1813
Illegal BglII site found at 533 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1813
Illegal PstI site found at 295
Illegal PstI site found at 304
Illegal PstI site found at 1456 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1813
Illegal PstI site found at 295
Illegal PstI site found at 304
Illegal PstI site found at 1456
Illegal AgeI site found at 1495 - 1000COMPATIBLE WITH RFC[1000]
| strain | E.coli BL21(DE3) |