Difference between revisions of "Part:BBa K4632002"

(Construction and Characterization)
 
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<partinfo>BBa_K4632002 short</partinfo>
 
<partinfo>BBa_K4632002 short</partinfo>
  
<h2>Description</h2>
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===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)
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<p>''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)</p>
  
  
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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)
 
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'''
 
'''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/model])
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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 [https://2023.igem.wiki/scau-china/model])
  
  
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'''3. 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.
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<p>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. </p>
  
      <p> 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.</p>
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<p>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 )</p>
  
      <p>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.</p>
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<p>'''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 )</p>
  
      <p>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.)</p>
 
  
<br>
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''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-7-1.png''
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<p><strong>Figure 1.</strong>Diagram of Cry3A-like Toxin circuit design</p>
  
</html>
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''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-20-1.png''
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<p><strong>Figure 2.</strong>pET30a-OmpA-Cry3A-like toxin with a 6×His tag</p>
  
<h2>Sequence and Features</h2>
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===Sequence and Features===
<partinfo>BBa_K4632002 SequenceAndFeatures</partinfo>
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----
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<!-- -->
  
<html>
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<partinfo>BBa_K4632012 SequenceAndFeatures</partinfo>
  
<br>
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===Construction and Characterization===
  
<h2>Construction and Characterization</h2>  
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<p><strong> 1. Construction</strong></p>  
  
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''https://static.igem.wiki/teams/4632/wiki/9999999999999.png''
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<p><strong>Figure 3.</strong>Colony PCR</p>
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<p>Lane 1-2: Digestion products of pET30a-OmpA-Cry3A-like toxin</p>
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''https://static.igem.wiki/teams/4632/wiki/1111111111111111.png''
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<p><strong>Figure 3.</strong>Enzymes digestion assay of pET30a-OmpA-Cry3A-like toxin</p>
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<p>Lane 1-2: PCR products of pET30a OmpA-Cry3A-like toxin</p>
  
<p><strong> 1. Verifying the Expression and Secretion Proficiency of Cry3A-like Toxin</strong></p>
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<p><strong> 2. Verifying the Expression and Excretion Proficiency of Cry3A-like Toxin</strong></p>  
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.
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<br>
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<p>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.</p>
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''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-21.png''
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<p><strong>Figure 4 </strong>SDS-PAGE Electrophoresis Detection of Cry3A-like Toxin Expression.</p><p> 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)</p>
  
<figure align="center">
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<p>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.</p>
<img
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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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<p>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.</p>
  
</figure>
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<p><strong> 2. Poisonous protein validation model</strong></p>
 +
<p><strong>(1). Introduction of toxic protein</strong></p>
 +
      <p>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)</p>
 +
      <p>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.</p>
  
<br>
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<p><strong>(2). Determination of ligand protein</strong></p>  
<br>
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<p>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.</p>
  
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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<p><strong>(3). Protein modeling and protein-protein docking</strong></p>
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<p>The homologous modeling of Cry3A like protein and cadherin-23 was performed using Swiss-model. The modeling results are as follows.</p>
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''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-3666.png''
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<p><strong>Figure 5</strong>cadherin-23 left, Cry3A like protein right, see Cry3A _ like _ protein.pdb, Cry3A _ like _ protein.stl, cadherin-23.pdb, cadherin-23.stl[https://2023.igem.wiki/scau-china/model]<p>
  
<br>
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    <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.</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.</p>
 +
''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-5-1-1.png''
  
<figure align="center">
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    <p>Among them, the hydrogen bonds and salt bridge sites formed by protein docking are shown in the following table.</p>
<img
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''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-6-2-1.png''
alt="parts1"
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    <p>The docking surface is shown in the following figure (see the docking.pdb, docking.x3d file[[https://2023.igem.wiki/scau-china/model]] ).</p>
src="https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-2-2.png"
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''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-1-666.png''
width="700"
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<p><strong>Figure 6</strong>The docking surface of Cry3A like protein and cadherin-23</p>
title="SDS-PAGE analysis of Cry3A-like toxin expression"
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      <p>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.</p>
  
<p> <figcaption><strong> Figure 2: </strong>SDS-PAGE analysis of Cry3A-like toxin expression </figcaption></p>  
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<p><strong>(4). Molecular dynamics simulation of protein-protein complex</strong></p>  
 +
<p>The dynamic simulation of the protein is shown in the section Existing problems and future work.[https://2023.igem.wiki/scau-china/model]</p>
  
</figure>
 
  
<br>
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<h2>References</h2>  
<br>
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<p>Lee A. Bulla, Jr.Mehmet Candas Formicidae (ant) control using <i>Bacillus thuringiensis</i> toxin  US 6,551,800B1[P]. 2003-04-22.</p>
 +
<p>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.</p>
  
<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>
 
<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>
 
<br>
 
  
 
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<!-- Uncomment this to enable Functional Parameter display
<figure align="center">
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===Functional Parameters===
<img
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<partinfo>BBa_K4632003 parameters</partinfo>
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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<br>
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<br>
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<p><strong>2. Poisonous protein validation model</strong></p>
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<p>(1). Determination of ligand protein</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>(2). Protein modeling and protein-protein docking</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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<br>
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<figure align="center">
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<img
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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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<br>
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<br>
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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.
+
<br>
+
 
+
 
+
<figure align="center">
+
<img
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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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<br>
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<br>
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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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<br>
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<figure align="center">
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<img
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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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<br>
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<br>
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The docking surface is shown in the following figure ( <p>see the docking.pdb, docking.x3d file <a href="https://2023.igem.wiki/scau-china/model" class="pr-0" target="_blank">(scau-china/model)</a>).
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<br>
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<figure align="center">
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<img
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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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<br>
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<br>
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<h2>References</h2>
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<p>[1] Lee A. Bulla, Jr.Mehmet Candas Formicidae (ant) control using Bacillus thuringiensis toxin  US 6,551,800B1[P]. 2003-04-22.</p>
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<p>[2] 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.</p>
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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 )


part-1-7-1.png

Figure 1.Diagram of Cry3A-like Toxin circuit design

part-1-20-1.png

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

Sequence and Features



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 142
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Construction and Characterization

1. Construction

9999999999999.png

Figure 3.Colony PCR

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

1111111111111111.png

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.

part-1-21.png

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.

part-1-3666.png

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.

part-1-5-1-1.png

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

part-1-6-2-1.png

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

part-1-666.png

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.