Difference between revisions of "Part:BBa K3771027"

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−	<br>We ligased the <i>P<sub>lacI</sub>-ompA/oprF</i> fragment and <i>P<sub>pspA</sub>-jju</i> on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of JJU 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>-jju-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 JJU expression by western blot using the anti-6X his-tag antibody.
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	<img src="https://2021.igem.org/wiki/images/f/f3/T--NCKU_Tainan--pspAjjuv.jpg" style="width:40%;">		<img src="https://2021.igem.org/wiki/images/f/f3/T--NCKU_Tainan--pspAjjuv.jpg" style="width:40%;">
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−	<p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-jju</i></p>	+	<p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-jju-6xHis</i></p>
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Revision as of 20:50, 19 October 2021

PpspA-JJU-6xHis-PlacI-OmpA/OprF

Description

This composite part is a component of the IFN-γ sensing system and is used to express the taurine production enzyme, JJU.

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 JJU. JJU converts L-cysteine into L-cystate in the taurine synthesis L-cystate pathway.

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-jju-6xHis on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of JJU expression by western blot using the anti-6X his-tag antibody.

Characterization

Fig. 2. Double digestion check of P_pspA-jju-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/

Sequence and Features