Difference between revisions of "Part:BBa K3771018"
 
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<partinfo>BBa_K3771018 short</partinfo>
 
<partinfo>BBa_K3771018 short</partinfo>
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<br><b style="font-size:1.3rem">Description</b>
 
<br><b style="font-size:1.3rem">Description</b>
 
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<br>This composite part is part of a plasmid used in the IFN-γ induction assay to test the function of the IFN-γ sensing system. It consists of the ompA/oprF gene regulated by the ompA promoter (PompA-ompA/oprF) and the orange fluorescent protein (OFP) gene regulated by the pspA promoter (PpspA-ofp).<br>
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<br>This composite part is part of a plasmid used in the IFN-γ induction assay to test the function of the IFN-γ sensing system. It consists of the <i>ompA/oprF</i> gene regulated by the <i>ompA</i> promoter (<i>PompA-ompA/oprF</i>) and the orange fluorescent protein (OFP) gene regulated by the <i>pspA</i> promoter (<i>PpspA-ofp</i>).<br>
  
 
<br><b style="font-size:1.3rem">Biology</b>
 
<br><b style="font-size:1.3rem">Biology</b>
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<img src="https://2021.igem.org/wiki/images/a/af/T--NCKU_Tainan--ompA_oprF.gif" style="width: 35%">
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<img src="https://2021.igem.org/wiki/images/a/af/T--NCKU_Tainan--ompA_oprF.gif" style="width: 50%">
 
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<p align="center">Figure. 1 Structure of OmpA/OprF chimeric protein</p>
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<p align="center">Fig. 1. Structure of OmpA/OprF chimeric protein</p>
  
  
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<br>To allow <i>E. coli</i> to detect IFN-γ in the human gut, we constructed an OmpA/OprF chimeric protein designed by Aurand and March[1]. The OmpA/OprF chimeric protein consists of OmpA protein from <i>E. coli</i> and a smaller section of OprF protein from <i>P. aeruginosa</i>.<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/e/e6/T--NCKU_Tainan--pspA_ofp.gif
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" style="width:65%;">
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<p align="center">Fig. 2. Mechanism of IFN-γ sensing system</p>
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<br>The <i>ompA</i> promoter facilitates the constitutive expression of chimeric OmpA/OprF. Binding of IFN-γ to chimeric OmpA/OprF induces the response of the phage shock protein (Psp) system[1], a highly conserved stress response system in enterobacteria[2]. 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>For the IFN-γ induction assay, OFP is used as the reporter to compare the strengths of the <i>lacI</i> promoter (<a href="https://parts.igem.org/Part:BBa_R0010" alt="" target="_blank">BBa_R0010</a>) and the <i>ompA</i> promoter (<a href="https://parts.igem.org/Part:BBa_K3771011" alt="" target="_blank">BBa_K3771011</a>). Therefore, two plasmids with the respective promoters were constructed, while also containing the <i>pspA</i> promoter (<a href="https://parts.igem.org/Part:BBa_K3071013" alt="" target="_blank">BBa_K3071013</a>) required for the activation of the IFN-γ sensing system.
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First, the <i>pspA</i> promoter synthesized by IDT was amplified by PCR and then ligated to ofp gene (<a href="https://parts.igem.org/Part:BBa_K156009" alt="" target="_blank">BBa_K156009</a>), already present in our starting pUC cloning vector, resulting in <i>P<sub>pspA</sub>-ofp</i> (<a href="https://parts.igem.org/Part:BBa_K3771014" alt="" target="_blank">BBa_K3771014</a>). To construct <i>P<sub>lacI</sub>-ompA/oprF</i> (<a href="https://parts.igem.org/Part:BBa_K3771015" alt="" target="_blank">BBa_K3771015</a>), <i>lacI</i> promoter in our starting pUC cloning vector, was ligated with the <i>ompA/oprF</i> fragment (<a href="https://parts.igem.org/Part:BBa_K3771009" alt="" target="_blank">BBa_K3771009</a>) synthesized by IDT. We then cloned the <i>ompA</i> promoter sequence from <i>E. coli</i> MG1655 and replaced <i>lacI</i> promoter with <i>ompA</i> promoter, resulting in <i>P<sub>ompA</sub>-ompA/oprF</i> (<a href="https://parts.igem.org/Part:BBa_K3771016" alt="" target="_blank">BBa_K3771016</a>). <br> 
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  <br>Both the <i>P<sub>lacI</sub>-ompA/oprF</i> and <i>P<sub>ompA</sub>-ompA/oprF</i> fragments were double digested and ligated to the <i>P<sub>pspA</sub>-ofp</i> vector, respectively, which formed the final plasmids required for the IFN-γ assay: <i>P<sub>pspA</sub>-ofp-P<sub>lacI</sub>-ompA/oprF</i> and <i>P<sub>pspA</sub>-ofp-P<sub>ompA-ompA/oprF</i> plasmids.<br>
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<br><b style="font-size:1.3rem">Characterization</b>
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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/2/20/T--NCKU_Tainan--composite_pspA_OFP_pOmpA_ompA_oprF.jpg" style="width:50%;">
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<p align="center">Fig. 3. Confirmation of construction by PCR. M: Marker; Lane 1: <i>pspA</i> promoter (~218bp); Lane 2: <i>ompA</i> promoter (~309 bp); Lane 3: <i>ompA/oprF</i> (~1110bp)</p>
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<br>Human IFN-γ of concentrations 300 pM and 600 pM was added to bacteria culture to induce the system. OFP expression (RFU) was recorded using a microplate reader and normalized by OD<sub>600</sub>. As shown in Fig. 4, starting at around 12 hours after induction, OFP expression increased dramatically. <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/a/a3/T--NCKU_Tainan--proof_of_concept_5.jpg" style="width:50%;">
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  <p align="center">Fig. 4. OFP expression of <i>P<sub>pspA</sub>-ofp-P<sub>ompA</sub>-ompA/oprF</i> </p> 
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<br>As presented in Fig. 5, at 12 hours after induction, sample 2 (<i>P<sub>pspA</sub>-ofp-P<sub>ompA</sub>-ompA/oprF</i>) with the addition of 600 pM IFN-γ had higher OFP expression than that of sample 1 (<i>P<sub>pspA</sub>-ofp-P<sub>lacI</sub>-ompA/oprF</i>). Since <i>ompA</i> promoter stimulates higher protein expression than <i>lacI</i> promoter, we decided to incorporate the <i>ompA</i> promoter in our final biobrick to regulate the expression of <i>cs</i>.<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/5/5a/T--NCKU_Tainan--proof_of_concept_45.jpg" style="width:50%;">
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<p align="center">Fig. 5. OFP expression of <i>P<sub>pspA</sub>-ofp-P<sub>lacI</sub>-ompA/oprF</i> (sample 1) and OFP expression of <i>P<sub>pspA</sub>-ofp-P<sub>ompA</sub>-ompA/oprF</i> (sample 2) at 12 hours after IFN-γ induction</p> 
  
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<br><b style="font-size:1.3rem">References</b>
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<br>1. 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>2. Darwin AJ. The phage-shock-protein response. Molecular Microbiology. 2005;57(3):621-628. doi:10.1111/j.1365-2958.2005.04694.x<br> 
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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 02:34, 22 October 2021


PpspA-OFP-PompA-OmpA/OprF

Description

This composite part is part of a plasmid used in the IFN-γ induction assay to test the function of the IFN-γ sensing system. It consists of the ompA/oprF gene regulated by the ompA promoter (PompA-ompA/oprF) and the orange fluorescent protein (OFP) gene regulated by the pspA promoter (PpspA-ofp).

Biology

Fig. 1. Structure of OmpA/OprF chimeric protein


To allow E. coli to detect IFN-γ in the human gut, we constructed an OmpA/OprF chimeric protein designed by Aurand and March[1]. The OmpA/OprF chimeric protein consists of OmpA protein from E. coli and a smaller section of OprF protein from P. aeruginosa.

Fig. 2. Mechanism of IFN-γ sensing system


The ompA promoter facilitates the constitutive expression of chimeric OmpA/OprF. Binding of IFN-γ to chimeric OmpA/OprF induces the response of the phage shock protein (Psp) system[1], a highly conserved stress response system in enterobacteria[2]. Signal transduction from the outer membrane to the inner membrane activates the pspA promoter, initiating expression of OFP.

Usage

For the IFN-γ induction assay, OFP is used as the reporter to compare the strengths of the lacI promoter (BBa_R0010) and the ompA promoter (BBa_K3771011). Therefore, two plasmids with the respective promoters were constructed, while also containing the pspA promoter (BBa_K3071013) required for the activation of the IFN-γ sensing system. First, the pspA promoter synthesized by IDT was amplified by PCR and then ligated to ofp gene (BBa_K156009), already present in our starting pUC cloning vector, resulting in PpspA-ofp (BBa_K3771014). To construct PlacI-ompA/oprF (BBa_K3771015), lacI promoter in our starting pUC cloning vector, was ligated with the ompA/oprF fragment (BBa_K3771009) synthesized by IDT. We then cloned the ompA promoter sequence from E. coli MG1655 and replaced lacI promoter with ompA promoter, resulting in PompA-ompA/oprF (BBa_K3771016).

Both the PlacI-ompA/oprF and PompA-ompA/oprF fragments were double digested and ligated to the PpspA-ofp vector, respectively, which formed the final plasmids required for the IFN-γ assay: PpspA-ofp-PlacI-ompA/oprF and PpspA-ofp-PompA-ompA/oprF plasmids.

Characterization

Fig. 3. Confirmation of construction by PCR. M: Marker; Lane 1: pspA promoter (~218bp); Lane 2: ompA promoter (~309 bp); Lane 3: ompA/oprF (~1110bp)


Human IFN-γ of concentrations 300 pM and 600 pM was added to bacteria culture to induce the system. OFP expression (RFU) was recorded using a microplate reader and normalized by OD600. As shown in Fig. 4, starting at around 12 hours after induction, OFP expression increased dramatically.

Fig. 4. OFP expression of PpspA-ofp-PompA-ompA/oprF


As presented in Fig. 5, at 12 hours after induction, sample 2 (PpspA-ofp-PompA-ompA/oprF) with the addition of 600 pM IFN-γ had higher OFP expression than that of sample 1 (PpspA-ofp-PlacI-ompA/oprF). Since ompA promoter stimulates higher protein expression than lacI promoter, we decided to incorporate the ompA promoter in our final biobrick to regulate the expression of cs.

Fig. 5. OFP expression of PpspA-ofp-PlacI-ompA/oprF (sample 1) and OFP expression of PpspA-ofp-PompA-ompA/oprF (sample 2) at 12 hours after IFN-γ induction


References

1. 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
2. Darwin AJ. The phage-shock-protein response. Molecular Microbiology. 2005;57(3):621-628. doi:10.1111/j.1365-2958.2005.04694.x
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 2158
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]