Difference between revisions of "Part:BBa K3771039"
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<br><b style="font-size:1.3rem">Usage</b><br> | <br><b style="font-size:1.3rem">Usage</b><br> | ||
<br>We ligased the <i>cs</i> fragment and <i>pspA</i> promoter on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. | <br>We ligased the <i>cs</i> fragment and <i>pspA</i> promoter on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. | ||
| − | The his-tag allows for confirmation of CS expression by western blot using the anti-6X his-tag antibody.<br><b style="font-size:1.3rem">Characterization</b><br> | + | The his-tag allows for confirmation of CS expression by western blot using the anti-6X his-tag antibody.<br><br><b style="font-size:1.3rem">Characterization</b><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="https://2021.igem.org/wiki/images/4/46/T--NCKU_Tainan--colony_csad.jpg" style="width:40%;"> | <img src="https://2021.igem.org/wiki/images/4/46/T--NCKU_Tainan--colony_csad.jpg" style="width:40%;"> | ||
Revision as of 21:46, 19 October 2021
PpspA-CS-6xHis
Description
This composite part is a component of the IFN-γ sensing system and was used to express the taurine production enzyme, L-cysteine sulfonic acid synthase (CS).
Biology
The ompA 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 CS-his-tag. CS converts o-phospho-l-serine into L-cysteine sulfonic acid in the taurine synthesis L-cysteine sulfinic acid pathway [2].
Fig.1 Taurine pathways in E. coli
Usage
We ligased the cs fragment and pspA promoter on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of CS expression by western blot using the anti-6X his-tag antibody.
Characterization
Fig. 2. Colony PCR confirmation of the construction
Reference
1. Darwin AJ. The phage-shock-protein response. Molecular Microbiology. 2005;57(3):621-628. doi:10.1111/j.1365-2958.2005.04694.xhttps://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.8b05093https://pubmed.ncbi.nlm.nih.gov/30516051/
Sequence and Features