Difference between revisions of "Part:BBa K3771031"

 
 
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<partinfo>BBa_K3771031 short</partinfo>
 
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<br><b style="font-size:1.3rem">Description</b>
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<br>This composite part is a component of the IFN-γ sensing system and was used to express the taurine production enzyme, CSAD.
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<br><b style="font-size:1.3rem">Biology</b>
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<br>The LacI 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>
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<br><b style="font-size:1.3rem">Usage</b>
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<br>We ligased the <i>P<sub>lacI</sub>-ompA/oprF</i> fragment and <i>P<sub>pspA</sub>-csad-6xHis</i> on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of CSAD expression by western blot using the anti-6X his-tag antibody.
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<br><b style="font-size:1.3rem">Characterization</b>
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<br>Double digestion results are shown in Figure 2.
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<img src="https://2021.igem.org/wiki/images/2/2c/T--NCKU_Tainan--pspAcsadhv.jpg" style="width:40%;">
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<p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-csad-6xHis</i></p>
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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/4/46/T--NCKU_Tainan--colony_pspAcsadhompA.jpg
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" style="width:40%;">
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<p align="center">Fig. 3. Colony PCR confirmation of the construction</p>
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<br><b style="font-size:1.3rem">References</b>
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<br>1. Darwin AJ. The phage-shock-protein response. <i>Molecular Microbiology</i>. 2005;57(3):621-628. doi:10.1111/j.1365-2958.2005.04694.x
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<a href="https://pubmed.ncbi.nlm.nih.gov/16045608/" alt="" target="_blank">https://pubmed.ncbi.nlm.nih.gov/16045608/</a>
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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
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<a href="https://pubmed.ncbi.nlm.nih.gov/30516051/" alt="" target="_blank">https://pubmed.ncbi.nlm.nih.gov/30516051/</a>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 03:18, 22 October 2021


PpspA-CSAD-6xHis-PlacI-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 LacI 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 PlacI-ompA/oprF fragment and PpspA-csad-6xHis on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of CSAD expression by western blot using the anti-6X his-tag antibody.

Characterization

Double digestion results are shown in Figure 2.

Fig. 2. Double digestion check of PpspA-csad-6xHis

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 2691
  • 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