Difference between revisions of "Part:BBa K4632002"

Revision as of 16:45, 8 October 2023 (view source) LHL scau (Talk | contribs) ← Older edit		Revision as of 16:45, 8 October 2023 (view source) LHL scau (Talk | contribs) Newer edit →
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−	'''How does it work?'''	+	'''1. 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)		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)
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−	'''Eco-friendly and Safe'''	+	'''2. 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])		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])
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−	'''What we have done? (SCAU-China 2023)'''	+	'''3. 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.		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.

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Revision as of 16:45, 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)

1. 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)

2. 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 [1])

3. 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 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 and Characterization

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

We conducted PCR amplification on the plasmid (procured from GUANGZHOU IGE BIOTECHNOLOGY LTD) to obtain the OmpA - Cry3A-like toxin segment. Subsequently, we utilized the Gibson Assembly method with the ABclonal 2× MultiF Seamless Assembly Mix kit to clone this segment into the pET30(a) plasmid. This resulted in the creation of the plasmid pET30a-OmpA-Cry3A-like toxin, as depicted in plasmid map Figure 1. Finally, we performed a transformation of this plasmid into Escherichia coli BL21(DE3) to assess the secretion expression of the toxin protein.

The pET-30(a)-OmpA-Cry3A-like toxin (E.coli BL21(DE3)) was cultured overnight in LB medium. The following day, the culture was subjected to transformation, and IPTG induction was carried out for 3 hours. Subsequently, the culture was centrifuged at 6,000 rpm for 10 minutes to separate the cell pellet and the supernatant. Afterward, both fractions were subjected to SDS-PAGE analysis (as shown in F