Difference between revisions of "Part:BBa K4632002"

Latest revision as of 15:41, 12 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 from UTD-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, the coding gene fragment for the active Cry3A-like toxin will be transformed into E. coli by the pET-30a vector, to confer the ability to produce Cry3A-like toxin.

To enable its excretion, a signal peptide sequence, OmpA, was added to the N-terminal of the Cry3A-like toxin. OmpA is a well-studied signal peptide in E.coli for the secretion of foreign proteins. (Figure 1 )

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.(Figure 2 )

Figure 1.Diagram of Cry3A-like Toxin circuit design

Figure 2.pET30a-OmpA-Cry3A-like toxin with a 6×His tag

Sequence and Features

Construction and Characterization

1. Construction

Figure 3.Colony PCR

Lane 1-2: Digestion products of pET30a-OmpA-Cry3A-like toxin

Figure 3.Enzymes digestion assay of pET30a-OmpA-Cry3A-like toxin

Lane 1-2: PCR products of pET30a OmpA-Cry3A-like toxin

2. Verifying the Expression and Excretion 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 excretion 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 4 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 4). This indicates the successful excretion 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 excretion expression of Cry3A-like toxin.

2. Poisonous protein validation model

(1). Introduction of toxic protein

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 S. invicta. Cry3A-like toxins isolated from UTD-001 have been shown to be toxic to S.invicta.(Lee A. Bullaet al.,2003)

It has been shown that after treatment with papain in vitro, the Cry3A-like toxin prototoxin (73KD) forms an active toxin (67KD) that is toxic to S. invicta.

(2). Determination of ligand protein

We retrieved the amino acid sequence of the cadherin-like protein BT-R1 [ 1 ] from Manduca sexta in the NCBI database, which has been identified to interact with Bt Cry protein, and paired it with its homologous protein cadherin-23 in S.invicta using blast.

(3). Protein modeling and protein-protein docking

The homologous modeling of Cry3A like protein and cadherin-23 was performed using Swiss-model. The modeling results are as follows.

Figure 5cadherin-23 left, Cry3A like protein right, see Cry3A _ like _ protein.pdb, Cry3A _ like _ protein.stl, cadherin-23.pdb, cadherin-23.stl[2]<p> <p>We used GRAMM Global RAnge Molecular Matching ) to dock our toxic proteins and ligand proteins. PDBePISA was used to analyze the docking results. The docking mutual surface size was 2465.8, and the binding free energy was-4.2. The binding free energy was less than 0, and the docking was meaningful.

We used GRAMM Global RAnge Molecular Matching ) to dock our toxic proteins and ligand proteins. PDBePISA was used to analyze the docking results. The docking mutual surface size was 2465.8, and the binding free energy was-4.2. The binding free energy was less than 0, and the docking was meaningful.

Among them, the hydrogen bonds and salt bridge sites formed by protein docking are shown in the following table.

The docking surface is shown in the following figure (see the docking.pdb, docking.x3d file[[3]] ).

Figure 6The docking surface of Cry3A like protein and cadherin-23

The docking attitude binding free energy is less than 0, and the docking is meaningful. Our molecular docking simulat ion proves that the toxic protein we designed may bind to 23 cadherin-protein in the S.invicta and exert toxicity.

(4). Molecular dynamics simulation of protein-protein complex

The dynamic simulation of the protein is shown in the section Existing problems and future work.[4]

References

Lee A. Bulla, Jr.Mehmet Candas Formicidae (ant) control using Bacillus thuringiensis toxin US 6,551,800B1[P]. 2003-04-22.

Han, L., Zhao, K., & Zhang, J. (2009). Interaction between insect calreticulin and Bt Cry1A protein. Insect Knowledge, 2009(2), 7. DOI: CNKI:SUN:KCZS.0.2009-02-007.