Difference between revisions of "Part:BBa K3771031"

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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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−	<br>We ligased the <i>P<sub>lacI</sub>-ompA/oprF</i> fragment and <i>P<sub>pspA</sub>-csad</i> on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of CSAD expression by western blot using the anti-6X his-tag antibody.	+	<br>We ligased the <i>P<sub>lacI</sub>-ompA/oprF</i> fragment and <i>P<sub>pspA</sub>-csad-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 CSAD expression by western blot using the anti-6X his-tag antibody.
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−	<img src="https://2021.igem.org/wiki/images/d/d7/T--NCKU_Tainan--pspAcsadv.jpg" style="width:40%;">	+	<img src="https://2021.igem.org/wiki/images/2/2c/T--NCKU_Tainan--pspAcsadhv.jpg" style="width:40%;">
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−	<p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-csad</i></p>	+	<p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-csad-6xHis</i></p>
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−	<img src="https://2021.igem.org/wiki/images/2/22/T--NCKU_Tainan--colony_pspAcsadompA.jpg	+	<img src="https://2021.igem.org/wiki/images/4/46/T--NCKU_Tainan--colony_pspAcsadhompA.jpg
	" style="width:40%;">		" style="width:40%;">
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Latest revision as of 03:18, 22 October 2021

PpspA-CSAD-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, CSAD.

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 CSAD. CSAD catalyzes the decarboxylation of L-Cysteine sulfinic acid into hypotaurine, which is spontaneously oxidized to taurine[2].

Fig. 1. Taurine pathways in E. coli

Usage

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

Characterization

Double digestion results are shown in Figure 2.

Fig. 2. Double digestion check of P_pspA-csad-6xHis

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/

2. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093 https://pubmed.ncbi.nlm.nih.gov/30516051/
Sequence and Features