Difference between revisions of "Part:BBa K3771030"

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	<br>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 <i>pspA</i> promoter, initiating expression of CSAD. CSAD catalyzes the decarboxylation of L-Cysteine sulfinic acid into hypotaurine, which is spontaneously oxidized to taurine[2].		<br>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 <i>pspA</i> promoter, initiating expression of CSAD. CSAD catalyzes the decarboxylation of L-Cysteine sulfinic acid into hypotaurine, which is spontaneously oxidized to taurine[2].
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−		+	<div style="width=100%; display:flex; align-items: center; justify-content: center;">
		+	<img src="https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--taurine_pathway_1.png" style="width:60%;">
		+	</div>
		+	<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>
	<br>		<br>
−	<br>We ligased the pLacI-OmpA* fragment and pspA-CSAD on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid.	+	<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.
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−	<br>Double digestion results are shown in Figure 1.	+	<br>Double digestion results are shown in Fig. 2.
	<br>		<br>
	<div style="width=100%; display:flex; align-items: center; justify-content: center;">		<div style="width=100%; display:flex; align-items: center; justify-content: center;">
−	<img src="圖片網址" style="width:35%;">	+	<img src="https://2021.igem.org/wiki/images/d/d7/T--NCKU_Tainan--pspAcsadv.jpg" style="width:40%;">
	</div>		</div>
−	<p align="center">圖片描述</p>	+	<p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-csad</i></p>
−		+	<div style="width=100%; display:flex; align-items: center; justify-content: center;">
		+	<img src="https://2021.igem.org/wiki/images/2/22/T--NCKU_Tainan--colony_pspAcsadompA.jpg
		+	" style="width:40%;">
		+	</div>
		+	<p align="center">Fig. 3. Colony PCR confirmation of the construction</p>
	<br><b style="font-size:1.3rem">References</b>		<br><b style="font-size:1.3rem">References</b>
	<br>		<br>

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Latest revision as of 03:19, 22 October 2021

PpspA-CSAD-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 on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid.

Characterization

Double digestion results are shown in Fig. 2.

Fig. 2. Double digestion check of P_pspA-csad

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