Difference between revisions of "Part:BBa K5322003"

 
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==Usage and Biology==
 
==Usage and Biology==
 
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Plasm~~~~~~
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The plasmid pET29a-J23119-RBS-Mfp53-T7 utilizes the pET29a vector for high-level expression in <i>Escherichia coli</i>. This system is controlled by the strong constitutive promoter J23119, which regulates the expression of the mussel foot protein 5+mussel foot protein 3( Mfp53) . The ribosome binding site (RBS) ensures efficient translation of the mRNA, while the T7 terminator provides a clean and efficient termination for transcription. This system is designed for the effective expression of Mfp53 under conditions not influenced by environmental factors, allowing it to exhibit its adhesive properties.
 
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In o3119-``````````
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To express eukaryotic proteins using prokaryotic systems, we selected <i>Escherichia coli</i> BL21(DE3) as the host cell. To ensure efficient expression of the adhesive proteins, we employed the strong constitutive promoter J23119 as a regulatory element and used a protein linker (GGGGS) to connect Mfp3 and Mfp5. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-RBS-Mfp53-T7. Through homologous recombination, we integrated this plasmid into BL21(DE3) and selected individual bacterial colonies from several transformation plates for plasmid extraction. We performed PCR verification using specific primers targeting a 680 bp fragment, as illustrated in Figure 2-2. The plasmids with correctly localized bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-RBS-Mfp53-T7.
 
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<img src="https://static.igem.wiki/teams/5322/wet-lab/20-pet29a-j23119-rbs-mfp3-t7.png" alt="pET29a-J23119-RBS-Mfp3-T7" width="300">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/33-pet29a-j23119-rbs-mfp53-t7.png" alt="pET29a-J23119-RBS-Mfp53-T7" width="600">
<p align="center"><b>Figure 2-1</b>  Plasmid pET29(a)-p<i>J23119-SoxR</i>-T-p<i>SoxS</i>-RBS-<i>Mfp3</i>-T7</p>
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<p align="center"><b>Figure 2-1</b>  Plasmid pET29a-J23119-RBS-Mfp53-T7</p>
 
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<img src="https://static.igem.wiki/teams/5101/partpage/phix174e-gel.png" alt="gel" width="500">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/39-pcr-mfp13.png" alt="gel" width="600">
<p align="center"><b>Figure 2-2</b>  Colony PCR gel electrophoresis of plasmid pET29(a)-p<i>J23119-SoxR</i>-T-p<i>SoxS</i>-RBS-PhiX174E-T7(1320bp)</p>
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<p align="center"><b>Figure 2-2</b>  Colony PCR gel electrophoresis of plasmid pET29a-J23119-RBS-Mfp53-T7(680bp)</p>
 
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<img src="https://static.igem.wiki/teams/5101/partpage/phix174e.png" alt="cexu" width="600">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/40-cexu-mfp1.png" alt="cexu" width="600">
<p align="center"><b>Figure 2-3</b>  plasmid pET29(a)-p<i>J23119-SoxR</i>-T-p<i>SoxS</i>-RBS-PhiX174E-T7 sequencing result</p>
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<p align="center"><b>Figure 2-3</b>  plasmid pET29a-J23119-RBS-Mfp53-T7 sequencing result</p>
 
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==Protein Expression Validation==
 
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We verified the performance of the lysis module through dry experiments and plan to complete wet experimental validation of its lytic function in the future. Through literature review and numerical modeling validation, we found that after inducing the expression of antimicrobial peptides and lysis proteins with NO for twenty minutes, the engineered strain will be lysed by the lysis protein and release the antimicrobial peptides. Mathematical modeling confirmed that at this time, the concentration of antimicrobial peptides is sufficient to reach an effective inhibitory concentration.
 
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==Sequence and Features==
 
==Sequence and Features==
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==Functional Parameters==
 
==Functional Parameters==
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Latest revision as of 03:17, 2 October 2024

Constitutive Mfp53 Expression System

Usage and Biology

The plasmid pET29a-J23119-RBS-Mfp53-T7 utilizes the pET29a vector for high-level expression in Escherichia coli. This system is controlled by the strong constitutive promoter J23119, which regulates the expression of the mussel foot protein 5+mussel foot protein 3( Mfp53) . The ribosome binding site (RBS) ensures efficient translation of the mRNA, while the T7 terminator provides a clean and efficient termination for transcription. This system is designed for the effective expression of Mfp53 under conditions not influenced by environmental factors, allowing it to exhibit its adhesive properties.

Construction of the plasmid

To express eukaryotic proteins using prokaryotic systems, we selected Escherichia coli BL21(DE3) as the host cell. To ensure efficient expression of the adhesive proteins, we employed the strong constitutive promoter J23119 as a regulatory element and used a protein linker (GGGGS) to connect Mfp3 and Mfp5. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-RBS-Mfp53-T7. Through homologous recombination, we integrated this plasmid into BL21(DE3) and selected individual bacterial colonies from several transformation plates for plasmid extraction. We performed PCR verification using specific primers targeting a 680 bp fragment, as illustrated in Figure 2-2. The plasmids with correctly localized bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-RBS-Mfp53-T7.

pET29a-J23119-RBS-Mfp53-T7

Figure 2-1 Plasmid pET29a-J23119-RBS-Mfp53-T7

gel

Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29a-J23119-RBS-Mfp53-T7(680bp)

cexu

Figure 2-3 plasmid pET29a-J23119-RBS-Mfp53-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 487
    Illegal NotI site found at 451
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 137
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]