Part:BBa_K4632002

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, 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

Assembly Compatibility:

• 10
  INCOMPATIBLE 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
  
• 12
  INCOMPATIBLE 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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1813
  Illegal BglII site found at 533
  
• 23
  INCOMPATIBLE 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
  
• 25
  INCOMPATIBLE 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
  
• 1000
  COMPATIBLE WITH RFC[1000]

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 E. coli BL21(DE3) to assess the secretion expression of the toxin protein.

Figure 1: Construction of pET30a-OmpA-Cry3A-like toxin

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 Figure 2)

Figure 2: SDS-PAGE analysis of Cry3A-like toxin expression

The supernatant from the induced culture displayed a distinct 66.6 kDa band corresponding to Cry3A-like toxin, which was absent in the supernatant of the non-induced culture and the wild-type control. This observation confirms the successful secretion expression of Cry3A-like toxin.

Subsequently, we utilized the enzyme digestion ligation/ΩPCR method to add a 6×His tag to pET30a-OmpA-Cry3A-like toxin. This modification was carried out to facilitate Western blot experiments, further confirming the secretion expression of Cry3A-like toxin (as depicted in plasmid map Figure 3).

Figure 3: pET30a-OmpA-Cry3A-like toxin with a 6×His tag

2. Poisonous protein validation model

(1). Determination of ligand protein

We retrieved the amino acid sequence of the cadherin-like protein BT-R1 [ 2 ] 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.

(2). 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 ( cadherin-23 left, Cry3A like protein right,

see Cry3A _ like _ protein.pdb, Cry3A _ like _ protein.stl, cadherin-23.pdb, cadherin-23.stl (scau-china/model)).

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 [https://2023.igem.wiki/scau-china/model]).

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

Molecular dynamics simulation of protein-protein complexes was performed using GROMACS to verify docking stability. We simulate in a hexagonal water box and a full atomic force field. Before the simulation, energy minimization and 50 ps NVT balance are performed respectively. And NPT balance.

It can be seen from the following figure that the temperature of the system is quickly stabilized at about 300K, and it has been stabilized at about 300K before 10ps.

Figure 4: Molecular dynamics simulation of protein-protein complex

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.