Difference between revisions of "Part:BBa K3771016"

Latest revision as of 01:40, 22 October 2021

PompA-OmpA/OprF

Description

P_ompA-ompA/oprF is part of the IFN-γ sensing system. ompA promoter regulates the constitutive expression of OmpA/OprF.

Biology

In the IFN-γ sensing system, binding of IFN-γ to the OmpA/OprF chimeric protein activates the pspA promoter, producing the enzyme required for the synthesis of taurine.

Usage

The ompA promoter sequence was amplified from E. coli MG1655, digested, and purified. We ligased ompA promoter and the ompA/oprF gene expression vector pUC and transformed it into DH5α to complete construction of the plasmid. Then, the plasmid was purified and transformed into E. coli ompA mutant strain JW0940.

Characterization

Double digestion was done to confirm the construction.

Fig. 1. Double digestion check of P_ompA-ompA/oprF

Western blot was performed to confirm the expression of OmpA/OprF.

Fig. 2. Confirmation of protein expression by western blot. Lane 1: OmpA positive control (~35 kDa); Lane 2: negative control; Lane 3, 4: OmpA/OprF chimeric protein (~39kDa)

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1142
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 29: / Line 29: @@
 <p align="center">Fig. 1. Double digestion check of <i>P<sub>ompA</sub>-ompA/oprF</i></p>
-<br>Western blot was performed to confirm the expression of <i>OmpA/OprF</i>.<br>
+<br>Western blot was performed to confirm the expression of OmpA/OprF.<br>
 <div style="width=100%; display:flex; align-items: center; justify-content: center;">
 <img src="https://2021.igem.org/wiki/images/9/9b/T--NCKU_Tainan--results_ompA_wb.jpg" style="width:50%;">