Difference between revisions of "Part:BBa K3771027"
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| − | <br>This composite part is a component of the IFN-γ sensing system and is used to express the taurine production enzyme, | + | <br>This composite part is a component of the IFN-γ sensing system and is used to express the taurine production enzyme, JJU. |
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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 | + | <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 JJU. JJU converts L-cysteine into L-cystate in the taurine synthesis L-cystate pathway. |
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| − | + | <br>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. <a href="https://pubmed.ncbi.nlm.nih.gov/16045608/" alt="" target="_blank">[1]</a> 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 <a href="https://pubmed.ncbi.nlm.nih.gov/30516051/" alt="" target="_blank">[2]</a>. <br> | |
| + | <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> | ||
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| − | <br>We ligased the | + | <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. |
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<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/ | + | <img src="https://2021.igem.org/wiki/images/f/f3/T--NCKU_Tainan--pspAjjuv.jpg" style="width:40%;"> |
</div> | </div> | ||
| − | <p align="center"> | + | <p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-jju</i></p> |
<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/ | + | <img src="https://2021.igem.org/wiki/images/a/a8/T--NCKU_Tainan--colony_pspAjjuompA.jpg" style="width:40%;"> |
</div> | </div> | ||
| − | <p align="center"> | + | <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> | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Revision as of 20:49, 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 PlacI-ompA/oprF fragment and PpspA-jju 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 PpspA-jju
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
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 12
Illegal BamHI site found at 2382 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]