Difference between revisions of "Part:BBa K4632002:Design"
 
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C.What we have done?（SCAU-China 2023）
 
  
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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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__NOTOC__
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<partinfo>BBa_K4632002 short</partinfo>
  
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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 Escherichia coli for the secretion expression of foreign proteins. Our SDS-PAGE results confirmed the successful secretion expression of Cry3A-like toxin.
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<partinfo>BBa_K4632002 SequenceAndFeatures</partinfo>
  
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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.
 
  
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In addition, to validate the toxicity of the designed Cry3A-like toxin against red imported fire ants, we selected homologous receptors of Cry3A-like toxin known from NCBI in red imported fire ants. 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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<h2>Design Notes</h2>
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'''3. What we have done? (SCAU-China 2023)'''
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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>
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<p>To enable its secretion, 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>
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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>
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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>
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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>
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<h2>Source</h2>
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We obtained the sequence of the active Cry protoxin of UTD-001 from the original literature. After codon optimization, we entrusted GUANGZHOU IGE BIOTECHNOLOGY LTD to synthesize the gene fragment OmpA-active Cry protoxin of UTD-001.
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<h2>References</h2>
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<html>
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[1] Lee A. Bulla, Jr.Mehmet Candas Formicidae (ant) control using <i>Bacillus thuringiensis</i> toxin  US 6,551,800B1[P]. 2003-04-22.
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</html>

Latest revision as of 09:30, 11 October 2023


Cry3A-like toxin


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]


Design Notes

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 secretion, 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


Source

We obtained the sequence of the active Cry protoxin of UTD-001 from the original literature. After codon optimization, we entrusted GUANGZHOU IGE BIOTECHNOLOGY LTD to synthesize the gene fragment OmpA-active Cry protoxin of UTD-001.

References

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