Difference between revisions of "Part:BBa K5101002"

 
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<partinfo>BBa_K5101002 short</partinfo>
 
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This composite plasmid, pET29a-p<i>J23119-SoxR</i>-T-p<i>SoxS</i>-RBS-eGFP-RBS-AMP-T7, is engineered for precise expression control in response to oxidative stress conditions. It incorporates a dual-promoter system where the first part consists of a strong constitutive promoter <i>J23119</i> for consistent expression of eGFP, serving as a reporting system. Following the J23119, a nitric-oxide-inducible promoter system, SoxR/SoxS, is used to control the expression of an antimicrobial peptide (AMP), enabling specific activation under stress conditions. The plasmid is equipped with Ribosome Binding Sites (RBS) for optimal translation efficiency of both proteins, ensuring robust expression in <i>E. coli</i> Nissle 1917. The T7 terminator ensures transcriptional termination, maintaining system stability and efficiency.
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Nitric-oxide-inducible 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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This composite plasmid, pET29a-p<i>J23119-SoxR</i>-T-p<i>SoxS</i>-RBS-eGFP-RBS-AMP-T7, is engineered for precise expression control in response to oxidative stress conditions. It incorporates a dual-promoter system where the first part consists of a strong constitutive promoter <i>J23119</i> for consistent expression of eGFP, serving as a reporting system. Following the p<i>J23119</i>, a nitric-oxide-inducible promoter system, <i>SoxR/SoxS</i>, is used to control the expression of an antimicrobial peptide (AMP), enabling specific activation under stress conditions. The plasmid is equipped with Ribosome Binding Sites (RBS) for optimal translation efficiency of both proteins, ensuring robust expression in <i>E. coli</i> Nissle 1917. The T7 terminator ensures transcriptional termination, maintaining system stability and efficiency.
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==Construction of the plasmid==
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<p>We designed the plasmid pET29a-p<i>J23119</i>-<i>SoxR</i>-T-p<i>SoxS</i>-RBS-eGFP-RBS-AMP-T7 as shown in Figure 1, and using homologous recombination integration, we constructed the expression plasmid pET29a-p<i>J23119</i>-<i>SoxR</i>-T-p<i>SoxS</i>-RBS-eGFP-RBS-AMP-AMP-T7. We transformed it into DH5α and extracted the recombinant plasmid after picking several single colonies of <i>E. coli</i> on the transformed plates for inoculation. We performed PCR to verify that the primers were specific primers with a target fragment of 2019bp, and the results are shown in Figures 2. We sent the plasmids with correct band positions to GENEWIZ Co. for sequencing. As shown in the figure 3, the sequencing results were all correct, which verified that the recombinant plasmid pET29a-p<i>J23119</i>-<i>SoxR</i>-T-p<i>SoxS</i>-RBS-eGFP-RBS-AMP-T7 was successfully constructed.
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<img src="https://static.igem.wiki/teams/5101/partpage/pet29-a-pj23119-soxr-t-psoxs-rbs-egfp-rbs-amp-t7-map.png" alt="plasmid2" width="420">
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<p align="center"><b>Figure 1</b>  NO-inducible plasmid pET29(a)-p<i>J23119</i>-<i>SoxR</i>-T-p<i>SoxS</i>-RBS-eGFP-RBS-AMP-T7</p>
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<img src="https://static.igem.wiki/teams/5101/partpage/responsive-amp-gel.png" alt="jiao2" width="450">
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<p align="center"><b>Figure 2</b>    M：DL2000 DNA Marker(Vazyme)</p>
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<p align="center">plasmid pET29(a)-p<i>J23119</i>-<i>SoxR</i>-T-p<i>SoxS</i>-RBS-eGFP-RBS-AMP-T7(2019bp)</p>
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<img src="https://static.igem.wiki/teams/5101/partpage/responsive-co-result.png" alt="result2" width="450">
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<p align="center"><b>Figure 3</b>  plasmid pET29(a)-p<i>J23119</i>-<i>SoxR</i>-T-p<i>SoxS</i>-RBS-eGFP-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_K5101002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5101002 SequenceAndFeatures</partinfo>
  

Revision as of 08:36, 11 September 2024


Plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7

Nitric-oxide-inducible antimicrobial peptide expression plasmid

Usage and Biology

This composite plasmid, pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7, is engineered for precise expression control in response to oxidative stress conditions. It incorporates a dual-promoter system where the first part consists of a strong constitutive promoter J23119 for consistent expression of eGFP, serving as a reporting system. Following the pJ23119, a nitric-oxide-inducible promoter system, SoxR/SoxS, is used to control the expression of an antimicrobial peptide (AMP), enabling specific activation under stress conditions. The plasmid is equipped with Ribosome Binding Sites (RBS) for optimal translation efficiency of both proteins, ensuring robust expression in E. coli Nissle 1917. The T7 terminator ensures transcriptional termination, maintaining system stability and efficiency.

Construction of the plasmid

We designed the plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7 as shown in Figure 1, and using homologous recombination integration, we constructed the expression plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-AMP-T7. We transformed it into DH5α and extracted the recombinant plasmid after picking several single colonies of E. coli on the transformed plates for inoculation. We performed PCR to verify that the primers were specific primers with a target fragment of 2019bp, and the results are shown in Figures 2. We sent the plasmids with correct band positions to GENEWIZ Co. for sequencing. As shown in the figure 3, the sequencing results were all correct, which verified that the recombinant plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7 was successfully constructed.

plasmid2

Figure 1 NO-inducible plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7


jiao2

Figure 2 M：DL2000 DNA Marker(Vazyme)

plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7(2019bp)


result2

Figure 3 plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-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
    Illegal NheI site found at 572
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1708
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1678
  • 1000
    COMPATIBLE WITH RFC[1000]