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| − | ===Construction and Characterization===
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| − | <p><strong> 1. Verifying the Expression and Secretion Proficiency of Cry3A-like Toxin</strong></p>
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| − | 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 <i>E. coli</i> BL21(DE3) to assess the secretion expression of the toxin protein.
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| − | <br>
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| − | <figure align="center">
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| − | alt="parts1"
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| − | src="https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-7.png"
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| − | width="700"
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| − | title="Construction of pET30a-OmpA-Cry3A-like toxin"
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| − | <p> <figcaption><strong> Figure 1: </strong>Construction of pET30a-OmpA-Cry3A-like toxin </figcaption></p>
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| − | </figure>
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| − | <br>
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| − | 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)
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| − | <br>
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| − | <figure align="center">
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| − | alt="parts1"
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| − | src="https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-10.png"
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| − | width="700"
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| − | title="SDS-PAGE analysis of Cry3A-like toxin expression"
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| − | <p> <figcaption><strong> Figure 2: </strong>SDS-PAGE analysis of Cry3A-like toxin expression lane1:Concentrated protein supernatant of pET-30a(+IPTG); lane2:Concentrated protein supernatant of pET-30a-OmpA-Cry3A-like toxin(+IPTG); lane3:Concentrated protein supernatant of pET-30a-OmpA-Cry3A-like toxin(-IPTG) </figcaption></p>
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| − | </figure>
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| − | <p> 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.</p>
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| − | <p> 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).</p>
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| − | <br>
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| − | <figure align="center">
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| − | alt="parts1"
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| − | src="https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-1.png"
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| − | width="700"
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| − | title="Construction of pET30a-OmpA-Cry3A-like toxin with a 6×His tag"
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| − | <p> <figcaption><strong> Figure 3: </strong>pET30a-OmpA-Cry3A-like toxin with a 6×His tag </figcaption></p>
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| − | </figure>
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| − | <p><strong>2. Poisonous protein validation model</strong></p>
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| − | <p><strong>(1). Determination of ligand protein</strong></p>
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| − | 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.
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| − | <p><strong>(2). Protein modeling and protein-protein docking</strong></p>
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| − | 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, <p>see Cry3A _ like _ protein.pdb, Cry3A _ like _ protein.stl, cadherin-23.pdb, cadherin-23.stl <a href="https://2023.igem.wiki/scau-china/model" class="pr-0" target="_blank">(scau-china/model)</a>).
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| − | alt="parts1"
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| − | src="https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-3.png"
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| − | width="600"
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| − | </figure>
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| − | 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.
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| − | alt="parts1"
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| − | src="https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-5.png"
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| − | width="700"
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| − | </figure>
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| − | Among them, the hydrogen bonds and salt bridge sites formed by protein docking are shown in the following table.
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| − | <figure align="center">
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| − | alt="parts1"
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| − | src="https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-6.png"
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| − | width="700"
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| − | </figure>
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| − | The docking surface is shown in the following figure ( see the docking.pdb, docking.x3d file [https://2023.igem.wiki/scau-china/model]).
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| − | alt="parts1"
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| − | src="https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-4.png"
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| − | width="700"
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| − | </figure>
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UNIQcca50f1c7326f1b3-partinfo-00000000-QINU
UNIQcca50f1c7326f1b3-partinfo-00000001-QINU
