Difference between revisions of "Part:BBa K5322007"

 
 
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===Usage and Biology===
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==Usage and Biology==
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The plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7 utilizes the pET29a vector for high-level expression in <i>Escherichia coli</i>. To enhance intestinal mucosal repair, we employed the oxidative stress-responsive SoxR/SoxS promoter system, targeting treatment at the site of intestinal inflammation in a high nitric oxide (NO) environment. The expression of SoxR is controlled by the strong constitutive promoter J23119, which responds to NO and activates the SoxS promoter to regulate the expression of the mussel foot protein Mfp53. The ribosome binding site (RBS) ensures efficient translation of mRNA, while the T7 terminator provides a clean and efficient transcriptional end. This system is designed for the effective expression of Mfp53 in high NO environments associated with intestinal inflammation, facilitating its adhesive properties.
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==Construction of the plasmid==
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Due to the lack of endotoxin in <i>Escherichia coli</i> Nissle 1917 (EcN), we chose EcN as the chassis cell to enhance safety. To achieve efficient expression of adhesive proteins, we selected the strong promoter J23119 as the regulatory element and used the protein linker (GGGGS) to connect Mfp3 and Mfp5. To improve intestinal mucosal repair in a high nitric oxide (NO) environment associated with intestinal inflammation, we employed the oxidative stress-responsive SoxR/SoxS promoter system, targeting treatment at the site of intestinal inflammation. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7. Through homologous recombination, we transferred this plasmid into EcN, followed by the selection of single colonies from several transformation plates for plasmid extraction. We verified the presence of the target 1333 bp fragment using specific primers in a PCR assay, as depicted in Figure 2-2. Plasmids with correctly positioned bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7.
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<img src="https://static.igem.wiki/teams/5322/wet-lab/56-pet29a-j23119-soxr-t-psoxs-rbs-mfp53-t7.png"  alt="pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7"width="600">
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<p align="center"><b>Figure 2-1</b>  Plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7</p>
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<img src="https://static.igem.wiki/teams/5322/wet-lab/61-pcr-mfp28.png" alt="gel" width="600">
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<p align="center"><b>Figure 2-2</b>  Colony PCR gel electrophoresis of plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7(1333bp)</p>
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<img src="https://static.igem.wiki/teams/5322/wet-lab/62-cexu-mfp2.png" alt="cexu" width="600">
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<p align="center"><b>Figure 2-3</b>  plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7 sequencing result</p>
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==Sequence and Features==
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===Functional Parameters===
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==Functional Parameters==
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<partinfo>BBa_K5101007 parameters</partinfo>
 
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Latest revision as of 03:07, 2 October 2024

NO-inducible Mfp53 Expression System


Usage and Biology

The plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7 utilizes the pET29a vector for high-level expression in Escherichia coli. To enhance intestinal mucosal repair, we employed the oxidative stress-responsive SoxR/SoxS promoter system, targeting treatment at the site of intestinal inflammation in a high nitric oxide (NO) environment. The expression of SoxR is controlled by the strong constitutive promoter J23119, which responds to NO and activates the SoxS promoter to regulate the expression of the mussel foot protein Mfp53. The ribosome binding site (RBS) ensures efficient translation of mRNA, while the T7 terminator provides a clean and efficient transcriptional end. This system is designed for the effective expression of Mfp53 in high NO environments associated with intestinal inflammation, facilitating its adhesive properties.

Construction of the plasmid

Due to the lack of endotoxin in Escherichia coli Nissle 1917 (EcN), we chose EcN as the chassis cell to enhance safety. To achieve efficient expression of adhesive proteins, we selected the strong promoter J23119 as the regulatory element and used the protein linker (GGGGS) to connect Mfp3 and Mfp5. To improve intestinal mucosal repair in a high nitric oxide (NO) environment associated with intestinal inflammation, we employed the oxidative stress-responsive SoxR/SoxS promoter system, targeting treatment at the site of intestinal inflammation. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7. Through homologous recombination, we transferred this plasmid into EcN, followed by the selection of single colonies from several transformation plates for plasmid extraction. We verified the presence of the target 1333 bp fragment using specific primers in a PCR assay, as depicted in Figure 2-2. Plasmids with correctly positioned bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7.

pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7

Figure 2-1 Plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7

gel

Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7(1333bp)

cexu

Figure 2-3 plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7 sequencing result


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 603
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 603
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 603
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 603
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 603
    Illegal AgeI site found at 585
  • 1000
    COMPATIBLE WITH RFC[1000]