Difference between revisions of "Part:BBa K4632002"

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	Amplification of the OmpA - Cry3A-like toxin segment from the plasmid (procured from GUANGZHOU IGE BIOTECHNOLOGY LTD) was conducted via PCR. Subsequently, the fragment was cloned into the pET30(a) plasmid using Gibson Assembly, employing the ABclonal 2× MultiF Seamless Assembly Mix kit. This yielded the plasmid pET30a-OmpA-Cry3A-like toxin (as illustrated in plasmid map, Figure 1). Finally, this plasmid was transformed into Escherichia coli BL21(DE3).		Amplification of the OmpA - Cry3A-like toxin segment from the plasmid (procured from GUANGZHOU IGE BIOTECHNOLOGY LTD) was conducted via PCR. Subsequently, the fragment was cloned into the pET30(a) plasmid using Gibson Assembly, employing the ABclonal 2× MultiF Seamless Assembly Mix kit. This yielded the plasmid pET30a-OmpA-Cry3A-like toxin (as illustrated in plasmid map, Figure 1). Finally, this plasmid was transformed into Escherichia coli BL21(DE3).

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Revision as of 15:29, 8 October 2023

Cry3A-like toxin

Description

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)

How does it work?

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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)

Eco-friendly and Safe

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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 [1])

What we have done? (SCAU-China 2023)

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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 E. 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 S. Invicta, we selected homologous receptors of Cry3A-like toxin known from NCBI in S. Invicta. 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.)

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Sequence and Features

Construction

'''Verifying the Expression and Secretion Proficiency of Cry3A-like Toxin'''

---- Amplification of the OmpA - Cry3A-like toxin segment from the plasmid (procured from GUANGZHOU IGE BIOTECHNOLOGY LTD) was conducted via PCR. Subsequently, the fragment was cloned into the pET30(a) plasmid using Gibson Assembly, employing the ABclonal 2× MultiF Seamless Assembly Mix kit. This yielded the plasmid pET30a-OmpA-Cry3A-like toxin (as illustrated in plasmid map, Figure 1). Finally, this plasmid was transformed into Escherichia coli BL21(DE3). Sequence and Features BBa_K4632002 SequenceAndFeatures