Difference between revisions of "Part:BBa K3771014"

 
 
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<br><b style="font-size:1.3rem">Description</b>
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<br>
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<br><i>P<sub>pspA</sub>-ofp</i> is part of the IFN-γ sensing system and was used in the IFN-γ induction assay, testing the efficiency of the <i>pspA</i> promoter.<br>
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<br><b style="font-size:1.3rem">Biology</b>
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<br>
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<br>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 OFP.<br>
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<br><b style="font-size:1.3rem">Usage</b>
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<br>
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<br>We ligased the <i>pspA</i> promoter fragment and OFP on the pSU expression vector and transformed the plasmid into DH5α.<br>
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<br><b style="font-size:1.3rem">Characterization</b>
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<br>
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<br>Double digestion results are shown in Fig. 1.<br>
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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/f/f4/T--NCKU_Tainan--PpspA-ofp.jpg" style="width:35%;">
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<p align="center">Fig. 1. Confirmation of construction by double digestion. M: Marker; Lane 1: <i>P<sub>pspA</sub>-ofp</i></p>
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<br>To test the function of the <i>pspA</i> promoter, OFP expression regulated by the induction of the <i>pspA</i> promoter was observed and recorded using a microplate reader.<br>
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<br>
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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/2/24/T--NCKU_Tainan--elisa4.jpg" style="width:50%;">
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</div>
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  <p align="center">Fig. 2. OFP expression of <i>P<sub>pspA</sub>-ofp-P<sub>lacI</sub>-ompA/oprF</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/c/c0/T--NCKU_Tainan--elisa5.jpg" style="width:50%;">
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</div>
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<p align="center">Fig. 3. OFP expression of <i>P<sub>pspA</sub>-ofp-P<sub>ompA</sub>-ompA/oprF</i></p>
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<br><b style="font-size:1.3rem">Reference</b>
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<br>
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<br>1. Darwin AJ. The phage-shock-protein response. Molecular Microbiology. 2005;57(3):621-628. doi:10.1111/j.1365-2958.2005.04694.x
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https://pubmed.ncbi.nlm.nih.gov/16045608/
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<br>2. Aurand TC, March JC. Development of a synthetic receptor protein for sensing inflammatory mediators interferon‐γ and tumor necrosis factor‐α. Biotechnology and Bioengineering. 2016;113(3):492-500. doi:10.1002/bit.25832
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<br>3. Manganelli R, Gennaro ML. Protecting from Envelope Stress: Variations on the Phage-Shock-Protein Theme. Trends in Microbiology. 2017;25(3):205-216. doi:10.1016/j.tim.2016.10.001
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551406/
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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 03:23, 22 October 2021


PpspA-OFP


Description

PpspA-ofp is part of the IFN-γ sensing system and was used in the IFN-γ induction assay, testing the efficiency of the pspA promoter.

Biology

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 OFP.

Usage

We ligased the pspA promoter fragment and OFP on the pSU expression vector and transformed the plasmid into DH5α.

Characterization

Double digestion results are shown in Fig. 1.

Fig. 1. Confirmation of construction by double digestion. M: Marker; Lane 1: PpspA-ofp


To test the function of the pspA promoter, OFP expression regulated by the induction of the pspA promoter was observed and recorded using a microplate reader.

Fig. 2. OFP expression of PpspA-ofp-PlacI-ompA/oprF

Fig. 3. OFP expression of PpspA-ofp-PompA-ompA/oprF


Reference

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. Aurand TC, March JC. Development of a synthetic receptor protein for sensing inflammatory mediators interferon‐γ and tumor necrosis factor‐α. Biotechnology and Bioengineering. 2016;113(3):492-500. doi:10.1002/bit.25832
3. Manganelli R, Gennaro ML. Protecting from Envelope Stress: Variations on the Phage-Shock-Protein Theme. Trends in Microbiology. 2017;25(3):205-216. doi:10.1016/j.tim.2016.10.001 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551406/

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
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]