Difference between revisions of "Part:BBa K5101001"

 
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<partinfo>BBa_K5101001 short</partinfo>
 
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Plasmid pET29a-pJ23119-RBS-AMP-T7 is designed for robust expression of a novel antimicrobial peptide (AMP) in Escherichia coli BL21(DE3). This plasmid incorporates the strong constitutive promoter J23119 to drive the expression of AMP, aimed at providing a high-level output suitable for therapeutic applications. The RBS enhances the initiation of translation, ensuring efficient protein synthesis. The T7 terminator ensures proper transcriptional termination. This part has been constructed to study the antimicrobial effects of the peptide in vitro, with potential applications in treating bacterial infections in animal models.
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Antimicrobial peptide expression plasmid
  
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===Usage and Biology===
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__TOC__
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==Usage and Biology==
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Plasmid pET29a-p<i>J23119</i>-RBS-AMP-T7 is designed for robust expression of a antimicrobial peptide(<partinfo>BBa_K5101000</partinfo>) in <i>Escherichia coli</i> BL21(DE3). This plasmid incorporates the strong constitutive promoter <i>J23119</i> to drive the expression of AMP, aimed at providing a high-level output suitable for therapeutic applications. The RBS enhances the initiation of translation, ensuring efficient protein synthesis. The T7 terminator ensures proper transcriptional termination. This part has been constructed to study the antimicrobial effects of the peptide in vitro, with potential applications in treating bacterial infections in animal models.
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==Construction of the plasmid==
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To express a antimicrobial peptide(</html><partinfo>BBa_K5101000</partinfo><html>), we selected <i>Escherichia coli</i> BL21 (DE3) and strong constitutive promoter <i>J23119</i>(</html><partinfo>BBa_J23119</partinfo><html>) as chassis cell and promoter to enable long-term and efficient antimicrobial peptide expression.
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Based on these design principles, we designed plasmid pET29a-p<i>J23119</i>-RBS-AMP-T7, as shown in Figure 1. Using homologous recombination integration, expression plasmid pET29a-p<i>J23119</i>-RBS-AMP-T7 was constructed and transformed into DH5α. We picked several single colonies of <i>E. coli</i> on the transfected plates and then extracted the recombinant plasmid and performed PCR to verify that the primers were specific and the target fragment was 587bp, and the results are shown in Figures 2. We sent the plasmids with the correct positions of the bands to GENEWIZ Co. for sequencing. As shown in the figure 3, the results of the sequencing were correct. The recombinant plasmid pET29a-p<i>J23119</i>-RBS-AMP-T7 was successfully constructed.
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<img src="https://static.igem.wiki/teams/5101/partpage/pet29-a-pj23119-rbs-amp-t7-map.png" alt="plasmid1" width="300">
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<p align="center"><b>Figure 1</b>  AMP expression plasmid pET29(a)-p<i>J23119</i>-RBS-AMP-T</p>
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<img src="https://static.igem.wiki/teams/5101/partpage/composive-amp-gel2.png" alt="jiao1" width="450">
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<p align="center"><b>Figure 2</b>    M：DL5000 DNA Marker(Vazyme)</p>
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<p align="center">plasmid pET29a-p<i>J23119</i>-RBS-AMP-T7(587bp)</p>
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<img src="https://static.igem.wiki/teams/5101/partpage/composive-co-result.png" alt="result1" width="450">
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<p align="center"><b>Figure 3</b>  plasmid pET29a-p<i>J23119</i>-RBS-AMP-T7 sequencing result</p>
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<span class='h3bb'>Sequence and Features</span>
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==Sequence and Features==
 
<partinfo>BBa_K5101001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5101001 SequenceAndFeatures</partinfo>
  

Latest revision as of 23:51, 24 September 2024


Plasmid pET29a-pJ23119-RBS-AMP-T7

Antimicrobial peptide expression plasmid

Usage and Biology

Plasmid pET29a-pJ23119-RBS-AMP-T7 is designed for robust expression of a antimicrobial peptide(BBa_K5101000) in Escherichia coli BL21(DE3). This plasmid incorporates the strong constitutive promoter J23119 to drive the expression of AMP, aimed at providing a high-level output suitable for therapeutic applications. The RBS enhances the initiation of translation, ensuring efficient protein synthesis. The T7 terminator ensures proper transcriptional termination. This part has been constructed to study the antimicrobial effects of the peptide in vitro, with potential applications in treating bacterial infections in animal models.

Construction of the plasmid

To express a antimicrobial peptide(BBa_K5101000), we selected Escherichia coli BL21 (DE3) and strong constitutive promoter J23119(BBa_J23119) as chassis cell and promoter to enable long-term and efficient antimicrobial peptide expression.

Based on these design principles, we designed plasmid pET29a-pJ23119-RBS-AMP-T7, as shown in Figure 1. Using homologous recombination integration, expression plasmid pET29a-pJ23119-RBS-AMP-T7 was constructed and transformed into DH5α. We picked several single colonies of E. coli on the transfected plates and then extracted the recombinant plasmid and performed PCR to verify that the primers were specific and the target fragment was 587bp, and the results are shown in Figures 2. We sent the plasmids with the correct positions of the bands to GENEWIZ Co. for sequencing. As shown in the figure 3, the results of the sequencing were correct. The recombinant plasmid pET29a-pJ23119-RBS-AMP-T7 was successfully constructed.

plasmid1

Figure 1 AMP expression plasmid pET29(a)-pJ23119-RBS-AMP-T


jiao1

Figure 2 M：DL5000 DNA Marker(Vazyme)

plasmid pET29a-pJ23119-RBS-AMP-T7(587bp)


result1

Figure 3 plasmid pET29a-pJ23119-RBS-AMP-T7 sequencing result


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 293
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 263
  • 1000
    COMPATIBLE WITH RFC[1000]