Difference between revisions of "Part:BBa K3771029"
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<br>We ligased the pLacI-OmpA* fragment and pspA-CoaBC-his-tag on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of CoaBC expression by western blot using the anti-6X his-tag antibody.
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<br>We ligased the <i>P<sub>lacI</sub>-ompA/oprF</i> fragment and <i>P<sub>pspA</sub>-coaBC-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 CoaBC expression by western blot using the anti-6X his-tag antibody.
 
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<img src="https://2021.igem.org/wiki/images/8/8b/T--NCKU_Tainan--digestion_pspA_CoaBC_6xHis_pLacI_ompA_oprF.jpg" style="width:35%;">
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<img src="https://2021.igem.org/wiki/images/1/1b/T--NCKU_Tainan--pspAcoav.jpg" style="width:35%;">
 
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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>-coaBC</i></p>
  
 
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<img src="https://2021.igem.org/wiki/images/c/cc/T--NCKU_Tainan--colony_pcr_pspA_CoaBC_6xHis_pLacI_ompA_oprF.jpg" style="width:35%;">
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<img src="https://2021.igem.org/wiki/images/d/d7/T--NCKU_Tainan--colony_pspAcoaompA.jpg
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<p align="center">圖片描述</p>
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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>

Revision as of 21:03, 19 October 2021


PpspA-CoaBC-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, CoaBC.

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 CoaBC. CoaBC converts L-cystate into taurine in the taurine synthesis L-cystate pathway.

Usage

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

Characterization

Fig. 2. Double digestion check of PpspA-coaBC

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/
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 2430
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 727
  • 1000
    COMPATIBLE WITH RFC[1000]