Difference between revisions of "Part:BBa K3771018"

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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.		<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.
−	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 ompA 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>	+	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>
	<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>		<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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Revision as of 01:52, 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

Figure. 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.

Figure 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 P_pspA-ofp (BBa_K3771014). To construct P_lacI-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 P_ompA-ompA/oprF (BBa_K3771016).

Both the P_lacI-ompA/oprF and P_ompA-ompA/oprF fragments were double digested and ligated to the P_pspA-ofp vector, respectively, which formed the final plasmids required for the IFN-γ assay: P_pspA-ofp-P_lacI-ompA/oprF and P_pspA-ofp-P_{ompA-ompA/oprF} plasmids.

Characterization

Figure 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 figure 11, starting at around 12 hours after induction, OFP expression increased dramatically.

Figure 4. OFP expression of P_pspA-ofp-P_ompA-ompA/oprF

As presented in figure 5, at 12 hours after induction, sample 2 (P_pspA-ofp-P_ompA-ompA/oprF) with the addition of 600 pM IFN-γ had higher OFP expression than that of sample 1 (P_pspA-ofp-P_lacI-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.

Figure 5. OFP expression of P_pspA-ofp-P_lacI-ompA/oprF (sample 1) and OFP expression of P_pspA-ofp-P_ompA-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