Difference between revisions of "Part:BBa K5322000"
 
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<partinfo>BBa_K5322000 short</partinfo>
 
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Nitric-oxide-inducible lysis module
 
  
 
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==Usage and Biology==
 
==Usage and Biology==
 
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Plasmid pET29(a)-p<i>J23119-SoxR</i>-T-p<i>SoxS</i>-RBS-PhiX174E-T7 leverages the pET29a vector backbone for high-level expression in <i>Escherichia coli</i>. It includes an engineered regulatory system composed of the strong constitutive promoter <i>J23119</i> followed by the nitric oxide-inducible <i>SoxR/SoxS</i> system, controlling the expression of the lysis protein PhiX174E. This design allows the lysis protein to be expressed in response to elevated nitric oxide levels, a common indicator of inflammatory conditions in cellular environments. The ribosome binding site (RBS) ensures efficient translation of the mRNA, and the T7 terminator provides a clean and efficient end to transcription. This system is intended for use in synthetic biology applications where controlled cell lysis is necessary, such as in timed release of therapeutic compounds or biocontainment strategies.
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The plasmid pET29a-J23119-RBS-Mfp3-T7 utilizes the pET29a vector for high-level expression of the mussel foot protein 3 (Mfp3) in <i>Escherichia coli</i>. This system is controlled by the strong constitutive promoter J23119, which regulates Mfp3 expression. The ribosome binding site (RBS) ensures efficient translation of the mRNA, while the T7 terminator provides a clean and efficient transcriptional endpoint. This system is designed for the efficient expression of Mfp3 under stable environmental conditions, allowing it to exert its adhesive properties.
 
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In order to allow the strain to lysis at the appropriate time and release product extracellularly, we designed a lysis module based on the lysis protein PhiX174E (91aa). The lysis protein PhiX174E (91aa) is a protein encoded by the PhiX174 phage gene E. Studies have shown that the PhiX174E protein triggers cell lysis through membrane binding and oligomerization with the host cell, as well as proton-dependent steps. As shown in Figure 2-1, we designed the plasmid pET29(a)-p<i>J23119-SoxR</i>-T-p<i>SoxS</i>-RBS-PhiX174E-T7. Through homologous recombination integration, this plasmid was transferred to DH5α, and plasmid extraction was performed after picking <i>E. coli</i> single colonies on several transformation plates. We conducted PCR validation with specific primers, targeting a 1320bp fragment, as shown in Figure 2-2. Plasmids with correctly positioned bands were sent to GENEWIZ Co. for sequencing. The sequencing results in Figure 2-3 confirmed that the plasmid pET29(a)-p<i>J23119-SoxR</i>-T-p<i>SoxS</i>-RBS-PhiX174E-T7 was successfully constructed.
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To enable the expression of eukaryotic proteins in prokaryotes, we selected <i>Escherichia coli</i> BL21(DE3) as the host cell. For efficient expression of the adhesin, we chose the strong promoter J23119 as the regulatory element. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-RBS-Mfp3-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 422 bp fragment, as depicted in Figure 2-2. The plasmids with correctly sized bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-RBS-Mfp3-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/20-pet29a-j23119-rbs-mfp3-t7.png" alt="pET29a-J23119-RBS-Mfp3-T7" width="600">
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<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-pJ23119-RBS-Mfp3-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/92-pcr-3.png" alt="gel" width="600">
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<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-pJ23119-RBS-Mfp3-T7(422bp)</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/93-cexu-3.png" alt="cexu" width="600">
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<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> Sequencing results of plasmid pET29a-J23119-RBS-Mfp3-T7</p>
 
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==Protein Expression Validation==
 
==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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We characterized the protein using Tricine-SDS-PAGE and Western Blot experiments, confirming the expression of Mfp3, as shown in Figures 3-1 and 3-2. The expected size of J23119-Mfp3, including the His tag, is 6.5 kDa; however, the bands at this position appear faint or even absent, while bands around 12 kDa are more prominent. We speculate that Mfp3 may have formed dimers or oligomers during the expression process.
 
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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/30-sds-mfp2.png" alt="SDS-PAGE" width="600">
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<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 3-1</b>  Tricine-SDS-PAGE analysis of Mfp3</p>
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<img src="https://static.igem.wiki/teams/5322/wet-lab/32-wb-mfp2.png" alt="WB" width="600">
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<p align="center"><b>Figure 3-2</b>  Western Blot analysis of Mfp3</p>
 
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==Sequence and Features==
 
==Sequence and Features==
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<partinfo>BBa_K5101004 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K5322000 SequenceAndFeatures</partinfo>
  
  
 
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==Functional Parameters==
 
==Functional Parameters==
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<partinfo>BBa_K5101004 parameters</partinfo>
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<partinfo>BBa_K5322000 parameters</partinfo>
 
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Latest revision as of 03:07, 2 October 2024


Constitutive Mfp3 Expression System


Usage and Biology

The plasmid pET29a-J23119-RBS-Mfp3-T7 utilizes the pET29a vector for high-level expression of the mussel foot protein 3 (Mfp3) in Escherichia coli. This system is controlled by the strong constitutive promoter J23119, which regulates Mfp3 expression. The ribosome binding site (RBS) ensures efficient translation of the mRNA, while the T7 terminator provides a clean and efficient transcriptional endpoint. This system is designed for the efficient expression of Mfp3 under stable environmental conditions, allowing it to exert its adhesive properties.

Construction of the plasmid

To enable the expression of eukaryotic proteins in prokaryotes, we selected Escherichia coli BL21(DE3) as the host cell. For efficient expression of the adhesin, we chose the strong promoter J23119 as the regulatory element. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-RBS-Mfp3-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 422 bp fragment, as depicted in Figure 2-2. The plasmids with correctly sized bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-RBS-Mfp3-T7.

pET29a-J23119-RBS-Mfp3-T7

Figure 2-1 Plasmid pET29a-pJ23119-RBS-Mfp3-T7

gel

Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29a-pJ23119-RBS-Mfp3-T7(422bp)

cexu

Figure 2-3 Sequencing results of plasmid pET29a-J23119-RBS-Mfp3-T7

Protein Expression Validation

We characterized the protein using Tricine-SDS-PAGE and Western Blot experiments, confirming the expression of Mfp3, as shown in Figures 3-1 and 3-2. The expected size of J23119-Mfp3, including the His tag, is 6.5 kDa; however, the bands at this position appear faint or even absent, while bands around 12 kDa are more prominent. We speculate that Mfp3 may have formed dimers or oligomers during the expression process.

SDS-PAGE

Figure 3-1 Tricine-SDS-PAGE analysis of Mfp3

WB

Figure 3-2 Western Blot analysis of Mfp3

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 210
    Illegal NotI site found at 174
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]