Difference between revisions of "Part:BBa K3771029"

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	<img src="https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--taurine_pathway_1.png" style="width:60%;">		<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>	+	<p align="center">Fig. 1. Taurine pathways in <i>E. coli</i></p>
	<br><b style="font-size:1.3rem">Usage</b>		<br><b style="font-size:1.3rem">Usage</b>
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−	<img src="https://2021.igem.org/wiki/images/1/1b/T--NCKU_Tainan--pspAcoav.jpg" style="width:35%;">	+	<img src="https://2021.igem.org/wiki/images/1/1b/T--NCKU_Tainan--pspAcoav.jpg" style="width:40%;">
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	<p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-coaBC</i></p>		<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/d/d7/T--NCKU_Tainan--colony_pspAcoaompA.jpg		<img src="https://2021.igem.org/wiki/images/d/d7/T--NCKU_Tainan--colony_pspAcoaompA.jpg
−	" style="width:35%;">	+	" style="width:40%;">
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	<p align="center">Fig. 3. Colony PCR confirmation of the construction</p>		<p align="center">Fig. 3. Colony PCR confirmation of the construction</p>

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Latest revision as of 03:19, 22 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.

Fig. 1. Taurine pathways in E. coli

Usage

We ligased the P_lacI-ompA/oprF fragment and P_pspA-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 P_pspA-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