Difference between revisions of "Part:BBa K3771036"
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<br>The <i>ompA</i> promoter facilitates the constitutive expression of OmpA/OprF. Binding of IFN-γ to the OmpA/OprF chimeric protein induces the response of the phage shock protein (Psp) system, a highly conserved stress response system in enterobacteria[1]. Signal transduction from the outer membrane to the inner membrane activates the <i>pspA</i> promoter, initiating expression of CSAD. CSAD catalyzes the decarboxylation of L-Cysteine sulfinic acid into hypotaurine, which is spontaneously oxidized to taurine[2].
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  <br>The <i>ompA</i> promoter facilitates the constitutive expression of OmpA/OprF. Binding of IFN-γ to the OmpA/OprF chimeric protein induces the response of the phage shock protein (Psp) system, a highly conserved stress response system in enterobacteria[1]. Signal transduction from the outer membrane to the inner membrane activates the <i>pspA</i> promoter, initiating expression of CSAD. CSAD catalyzes the decarboxylation of L-Cysteine sulfinic acid into hypotaurine, which is spontaneously oxidized to taurine[2].
 
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  <div style="width=100%; display:flex; align-items: center; justify-content: center;">
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<img src="https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--taurine_pathway_1.png" style="width:60%;">
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<p align="center">Fig.1 Taurine pathways in <i>E. coli</i></p>
 
<br><b style="font-size:1.3rem">Usage</b>
 
<br><b style="font-size:1.3rem">Usage</b>
 
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<br>We ligased the NPO-OmpA* fragment and pspA-CSAD on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid.
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<br>We ligased the <i>P<sub>ompA</sub>-ompA/oprF</i> fragment and <i>P<sub>pspA</sub>-csad</i> on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid.
 
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<br>Double digestion results are shown in Figure 1.
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<br>Double digestion results are shown in Figure 2.
 
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<img src="圖片網址" style="width:35%;">
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<img src="https://2021.igem.org/wiki/images/d/d7/T--NCKU_Tainan--pspAcsadv.jpg" style="width:40%;">
 
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<p align="center">圖片描述</p>
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<p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-csad</i></p>
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<img src="https://2021.igem.org/wiki/images/2/22/T--NCKU_Tainan--colony_pspAcsadompA.jpg
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<p align="center">Fig. 3. Colony PCR confirmation of the construction</p>
 
<br><b style="font-size:1.3rem">References</b>
 
<br><b style="font-size:1.3rem">References</b>
 
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<a href="https://pubmed.ncbi.nlm.nih.gov/16045608/" alt="" target="_blank">https://pubmed.ncbi.nlm.nih.gov/16045608/</a>
 
<a href="https://pubmed.ncbi.nlm.nih.gov/16045608/" alt="" target="_blank">https://pubmed.ncbi.nlm.nih.gov/16045608/</a>
 
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<br>2. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. <i>Journal of Agricultural and Food Chemistry</i>. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093
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2. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. <i>Journal of Agricultural and Food Chemistry</i>. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093
 
<a href="https://pubmed.ncbi.nlm.nih.gov/30516051/" alt="" target="_blank">https://pubmed.ncbi.nlm.nih.gov/30516051/</a>
 
<a href="https://pubmed.ncbi.nlm.nih.gov/30516051/" alt="" target="_blank">https://pubmed.ncbi.nlm.nih.gov/30516051/</a>
 
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Revision as of 21:31, 19 October 2021


PpspA-CSAD-PompA-OmpA/OprF


Description

This composite part is a component of the IFN-γ sensing system and was used to express the taurine production enzyme, CSAD.

Biology

The ompA promoter facilitates the constitutive expression of OmpA/OprF. Binding of IFN-γ to the OmpA/OprF chimeric protein induces the response of the phage shock protein (Psp) system, a highly conserved stress response system in enterobacteria[1]. Signal transduction from the outer membrane to the inner membrane activates the pspA promoter, initiating expression of CSAD. CSAD catalyzes the decarboxylation of L-Cysteine sulfinic acid into hypotaurine, which is spontaneously oxidized to taurine[2].

Fig.1 Taurine pathways in E. coli


Usage

We ligased the PompA-ompA/oprF fragment and PpspA-csad on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid.

Characterization

Double digestion results are shown in Figure 2.

Fig. 2. Double digestion check of PpspA-csad

Fig. 3. Colony PCR confirmation of the construction


References

1. Darwin AJ. The phage-shock-protein response. Molecular Microbiology. 2005;57(3):621-628. doi:10.1111/j.1365-2958.2005.04694.x https://pubmed.ncbi.nlm.nih.gov/16045608/

2. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093 https://pubmed.ncbi.nlm.nih.gov/30516051/
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 12
    Illegal BamHI site found at 2776
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 394
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 179