Difference between revisions of "Part:BBa K3771030"
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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> | ||
<br> | <br> |
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].
![](https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--taurine_pathway_1.png)
Fig. 1. Taurine pathways in E. coli
Usage
We ligased the PlacI-ompA/oprF fragment and PpspA-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.
![](https://2021.igem.org/wiki/images/d/d7/T--NCKU_Tainan--pspAcsadv.jpg)
Fig. 2. Double digestion check of PpspA-csad
![](https://2021.igem.org/wiki/images/2/22/T--NCKU_Tainan--colony_pspAcsadompA.jpg
)
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 12
Illegal BamHI site found at 2673 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 394
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 179