Difference between revisions of "Part:BBa K3771037"

Revision as of 21:33, 19 October 2021

PpspA-CSAD-6xHis-PompA-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 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 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_ompA-ompA/oprF fragment and P_pspA-csad-6xHis on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of CSAD expression by western blot using the anti-6X his-tag antibody.

Characterization

Double digestion results are shown in Figure 2.

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

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 12
Illegal BamHI site found at 2794
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 394
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 179

@@ Line 13: / Line 13: @@
 <br>
-<br>The <i>ompA</i> 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 <i>ompA</i> 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>
+  <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>
-<br>We ligased the NPO-OmpA* fragment and pspA-CSAD on the pSU expression vector and transformed it into DH5alpha to complete construction of the plasmid.  The his-tag allows for confirmation of CSAD expression by western blot using the anti-6X his-tag antibody.
+<br>We ligased the <i>P<sub>ompA</sub>-ompA/oprF</i> fragment and <i>P<sub>pspA</sub>-csad-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 CSAD expression by western blot using the anti-6X his-tag antibody.
 <br>
@@ Line 25: / Line 28: @@
 <br>
-<br>Double digestion results are shown in Figure 1.
+<br>Double digestion results are shown in Figure 2.
 <br>
 <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/2/2c/T--NCKU_Tainan--pspAcsadhv.jpg" style="width:40%;">
 </div>
-<p align="center">圖片描述</p>
+<p align="center">Fig. 2. Double digestion check of <i>P<sub>pspA</sub>-csad-6xHis</i></p>
+<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_pspAcsadhompA.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>
@@ Line 39: / Line 46: @@
 <a href="https://pubmed.ncbi.nlm.nih.gov/16045608/" alt="" target="_blank">https://pubmed.ncbi.nlm.nih.gov/16045608/</a>
 <br>
-<br>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. <i>Journal of Agricultural and Food Chemistry</i>. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093
+<br>
+. 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. <i>Journal of Agricultural and Food Chemistry</i>. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093
 <a href="https://pubmed.ncbi.nlm.nih.gov/30516051/" alt="" target="_blank">https://pubmed.ncbi.nlm.nih.gov/30516051/</a>
 <br>