Difference between revisions of "Part:BBa K5103001"

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===Profile===
 
===Profile===
 
Name: PCG004 with Cry8Da  
 
Name: PCG004 with Cry8Da  
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<br>Base Pairs: 12661 bp  
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<br>Base Pairs: 11543 bp  
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<br>Origin: Add
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<br>Origin: Synthetic plasmid <sup>[1]</sup>
 
<br>Properties: High copy number <sup>[1]</sup>
 
<br>Properties: High copy number <sup>[1]</sup>
  
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It includes features for selective gene expression in a dropout region that normally contains green fluorescent protein (GFP) flanked by BsaI sites. In this case we replaced GFP with Cry8Da crystal toxin sequences from <i> Bacillus thuringiensis </i> (<i>Bt</i>). Upstream, a lacI sequence encodes the lac repressor and a lac operator (lacO) overlaps with the pGrac promoter. Using this plasmid allows us to induce the expression of Cry8Da when we want using IPTG. This allows transcription of Cry8Da by causing a conformational change in Lac, releasing it from lacO.
 
It includes features for selective gene expression in a dropout region that normally contains green fluorescent protein (GFP) flanked by BsaI sites. In this case we replaced GFP with Cry8Da crystal toxin sequences from <i> Bacillus thuringiensis </i> (<i>Bt</i>). Upstream, a lacI sequence encodes the lac repressor and a lac operator (lacO) overlaps with the pGrac promoter. Using this plasmid allows us to induce the expression of Cry8Da when we want using IPTG. This allows transcription of Cry8Da by causing a conformational change in Lac, releasing it from lacO.
 +
<br><br>
 +
This is a composite part that includes pCG004 (see [[Part:BBa_K5103005]]), a shuttle plasmid backbone designed for <i> E. coli </i>, with the <i> Cry8Da </i> gene (see [[Part:BBa_K5103000]]) inserted. The <i> Cry8Da </i> gene is under the control of an IPTG-inducible promoter, enabling regulation of protein expression.<sup>[2]</sup>
  
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This is a composite part (see <partinfo>BBa_K5103000</partinfo>)that includes pCG004, a shuttle plasmid backbone designed for <i> E. coli </i>, with the <i> Cry8Da </i> gene inserted. The <i> Cry8Da </i> gene is under the control of an IPTG-inducible promoter, enabling regulation of protein expression.
+
Research has shown that Cry8Da effectively kills insects in the Coleoptera order.<sup>[3]</sup> The protein works by binding to receptors in the insect's midgut, leading to pore formation and eventual cell death.<sup>[3-6]</sup> The toxic domain of the Cry8Da protein is thought to be its 54 kDa active fragment, which is generated through proteolytic cleavage.<sup>[4-6]</sup>
  
−
This composite part has been demonstrated to work effectively in <i> E. coli </i> DH5α, and the expression of Cry8Da is regulated using the IPTG-inducible promoter.
+
Cry8Da is activated through proteolytic cleavage, and the resulting fragments are essential for the toxin’s insecticidal action, with different domains responsible for binding to receptors (C-terminal) and pore formation (N-terminal) in the gut cell of the beetle.<sup>[6]</sup>
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This system can be used for producing the Cry8Da protein in <i> E. coli </i> for laboratory studies, protein purification, and potential applications in bioinsecticide development.
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−
 
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INSERT INFORMATION ABOUT THE HISTORY OF THE PART
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<html><img src = "https://static.igem.wiki/teams/5103/bba-k5103001-1.webp"></html>
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<b> Figure 1 </b>. Streak plate of PCG-Cry8Da transformant colonies grown on ampicillin selective media.
+
  
 +
<br>
 +
This composite part has been demonstrated to work effectively in <i>E. coli</i> DH5α, and the expression of Cry8Da is regulated using the IPTG-inducible promoter.
 +
This system can be used for producing the Cry8Da protein in <i>E. coli</i> for laboratory studies, protein purification, and potential applications in bioinsecticide development. Furthermore, the IPTG-inducible promoter enables specificity in pesticide and other industrial applications.<sup>[2]</sup>
 +
<br><br>
 +
Our Guelph team was the first iGEM team to mention the use of pCG004 in the registry in 2022<sup>[7]</sup>. This plasmid is derived from pHT01, to be an entry vector (“shuttle”) to replicate within both <i>E. coli</i> and <i>Bacillus subtilis</i> (<i>B. subtilis</i>) and transfers a plasmid transformed within <i>E. coli</i> to the final destination of <i>B. subtilis</i>.<sup>[1]</sup> Due to its “shuttle abilities”, it has potential applications within bioagriculture and biocontrol, where genes with insecticidal properties can be inserted into a <i>Bacillus</i> species in the pests’ environment.
 +
<br><br>
 +
<html><img src = "https://static.igem.wiki/teams/5103/bba-k5103001-1.webp" class="center" style="width:500px"></html>
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<br><b>Figure 1.</b> Streak plate of PCG-Cry8Da transformant colonies grown on ampicillin selective media.
 
<br><br>
 
<br><br>
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<html><img src = "https://static.igem.wiki/teams/5103/bba-k5103001-plasmid-map.webp"></html>
 
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<b> Figure 2</b>. PCG-CRY plasmid components were developed using SnapGene.
 
  
 
===References===
 
===References===
 
[1] Gilbert, C., Howarth, M., Harwood, C. R., & Ellis, T. (2017). Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. <i>ACS synthetic biology</i>, <i>6</i>(6), 957–967. https://doi.org/10.1021/acssynbio.6b00292
 
[1] Gilbert, C., Howarth, M., Harwood, C. R., & Ellis, T. (2017). Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. <i>ACS synthetic biology</i>, <i>6</i>(6), 957–967. https://doi.org/10.1021/acssynbio.6b00292
 +
<br>[2] Chu, P. T. B., Phan, T. T. P., Nguyen, T. T. T., Truong, T. T. T., Schumann, W., & Nguyen, H.D. (2023). Potent IPTG-inducible integrative expression vectors for production of recombinant proteins in Bacillus subtilis. <i>World Journal of Microbiology and Biotechnology</i>, 39, 143. https://doi.org/10.1007/s11274-023-03566-8
 +
<br>[3] Shu, C., Yan, G., Huang, S., Geng, Y., Soberón, M., Bravo, A., Geng, L., & Zhang, J. (2020). Characterization of two novel Bacillus thuringiensis Cry8 toxins reveals differential specificity of protoxins or activated toxins against Chrysomeloidea coleopteran superfamily. <i>Toxins</i>, <i>12</i>(10), 642. https://doi.org/10.3390/toxins12100642
 +
<br>[4] Bravo, A., Gill, S. S., & Soberón, M. (2007). Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 49(4), 423-435. https://doi.org/10.1016/j.toxicon.2006.11.022
 +
<br>[5] Yamaguchi, T., Bando, H., & Asano, S. (2013). Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica. Journal of Invertebrate Pathology, 113(2), 123-128. https://doi.org/10.1016/j.jip.2013.03.006
 +
<br>[6] Yamaguchi, T., Sahara, K., Bando, H., & Asano, S. (2010). Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle. Journal of Invertebrate Pathology, 105(3), 243-247. https://doi.org/10.1016/j.jip.2010.07.002
 +
<br>[7] Janneh, K. (2022). Registry of Standard Biological Parts. IGEM Guelph 2022. https://parts.igem.org/Part:BBa_K4321005

Latest revision as of 21:34, 1 October 2024


PCG004 with Cry8Da

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal XbaI site found at 11533
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal NheI site found at 6234
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal BglII site found at 66
    Illegal BglII site found at 3147
    Illegal XhoI site found at 3151
    Illegal XhoI site found at 11410
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal XbaI site found at 11533
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal XbaI site found at 11533
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
    Illegal AgeI site found at 9294
    Illegal AgeI site found at 10176
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 11524
    Illegal BsaI.rc site found at 7949
    Illegal BsaI.rc site found at 7962
    Illegal BsaI.rc site found at 11537
    Illegal SapI.rc site found at 1219
    Illegal SapI.rc site found at 5124


Profile

Name: PCG004 with Cry8Da
Base Pairs: 11543 bp
Origin: Synthetic plasmid [1]
Properties: High copy number [1]

Usage and Biology

It includes features for selective gene expression in a dropout region that normally contains green fluorescent protein (GFP) flanked by BsaI sites. In this case we replaced GFP with Cry8Da crystal toxin sequences from Bacillus thuringiensis (Bt). Upstream, a lacI sequence encodes the lac repressor and a lac operator (lacO) overlaps with the pGrac promoter. Using this plasmid allows us to induce the expression of Cry8Da when we want using IPTG. This allows transcription of Cry8Da by causing a conformational change in Lac, releasing it from lacO.

This is a composite part that includes pCG004 (see Part:BBa_K5103005), a shuttle plasmid backbone designed for E. coli , with the Cry8Da gene (see Part:BBa_K5103000) inserted. The Cry8Da gene is under the control of an IPTG-inducible promoter, enabling regulation of protein expression.[2]

Research has shown that Cry8Da effectively kills insects in the Coleoptera order.[3] The protein works by binding to receptors in the insect's midgut, leading to pore formation and eventual cell death.[3-6] The toxic domain of the Cry8Da protein is thought to be its 54 kDa active fragment, which is generated through proteolytic cleavage.[4-6]

Cry8Da is activated through proteolytic cleavage, and the resulting fragments are essential for the toxin’s insecticidal action, with different domains responsible for binding to receptors (C-terminal) and pore formation (N-terminal) in the gut cell of the beetle.[6]


This composite part has been demonstrated to work effectively in E. coli DH5α, and the expression of Cry8Da is regulated using the IPTG-inducible promoter. This system can be used for producing the Cry8Da protein in E. coli for laboratory studies, protein purification, and potential applications in bioinsecticide development. Furthermore, the IPTG-inducible promoter enables specificity in pesticide and other industrial applications.[2]

Our Guelph team was the first iGEM team to mention the use of pCG004 in the registry in 2022[7]. This plasmid is derived from pHT01, to be an entry vector (“shuttle”) to replicate within both E. coli and Bacillus subtilis (B. subtilis) and transfers a plasmid transformed within E. coli to the final destination of B. subtilis.[1] Due to its “shuttle abilities”, it has potential applications within bioagriculture and biocontrol, where genes with insecticidal properties can be inserted into a Bacillus species in the pests’ environment.


Figure 1. Streak plate of PCG-Cry8Da transformant colonies grown on ampicillin selective media.

References

[1] Gilbert, C., Howarth, M., Harwood, C. R., & Ellis, T. (2017). Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS synthetic biology, 6(6), 957–967. https://doi.org/10.1021/acssynbio.6b00292
[2] Chu, P. T. B., Phan, T. T. P., Nguyen, T. T. T., Truong, T. T. T., Schumann, W., & Nguyen, H.D. (2023). Potent IPTG-inducible integrative expression vectors for production of recombinant proteins in Bacillus subtilis. World Journal of Microbiology and Biotechnology, 39, 143. https://doi.org/10.1007/s11274-023-03566-8
[3] Shu, C., Yan, G., Huang, S., Geng, Y., Soberón, M., Bravo, A., Geng, L., & Zhang, J. (2020). Characterization of two novel Bacillus thuringiensis Cry8 toxins reveals differential specificity of protoxins or activated toxins against Chrysomeloidea coleopteran superfamily. Toxins, 12(10), 642. https://doi.org/10.3390/toxins12100642
[4] Bravo, A., Gill, S. S., & Soberón, M. (2007). Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 49(4), 423-435. https://doi.org/10.1016/j.toxicon.2006.11.022
[5] Yamaguchi, T., Bando, H., & Asano, S. (2013). Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica. Journal of Invertebrate Pathology, 113(2), 123-128. https://doi.org/10.1016/j.jip.2013.03.006
[6] Yamaguchi, T., Sahara, K., Bando, H., & Asano, S. (2010). Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle. Journal of Invertebrate Pathology, 105(3), 243-247. https://doi.org/10.1016/j.jip.2010.07.002
[7] Janneh, K. (2022). Registry of Standard Biological Parts. IGEM Guelph 2022. https://parts.igem.org/Part:BBa_K4321005