Difference between revisions of "Part:BBa K3771016"
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<partinfo>BBa_K3771016 short</partinfo> | <partinfo>BBa_K3771016 short</partinfo> | ||
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<br><b style="font-size:1.3rem">Description</b> | <br><b style="font-size:1.3rem">Description</b> | ||
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<br><b style="font-size:1.3rem">Biology</b> | <br><b style="font-size:1.3rem">Biology</b> | ||
− | <br> | + | <br><br> |
− | 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.<br> | + | In the IFN-γ sensing system, binding of IFN-γ to the OmpA/OprF chimeric protein activates the <i>pspA</i> promoter, producing the enzyme required for the synthesis of taurine.<br> |
<br><b style="font-size:1.3rem">Usage</b> | <br><b style="font-size:1.3rem">Usage</b> | ||
<br> | <br> | ||
− | <br>The <i>ompA</i> promoter sequence was amplified from <i>E. coli</i> MG1655, digested, and purified. We ligased <i>ompA</i> promoter and the <i>ompA/oprF</i> gene expression vector | + | <br>The <i>ompA</i> promoter sequence was amplified from <i>E. coli</i> MG1655, digested, and purified. We ligased <i>ompA</i> promoter and the <i>ompA/oprF</i> gene expression vector pUC and transformed it into DH5α to complete construction of the plasmid. Then, the plasmid was purified and transformed into <i>E. coli ompA</i> mutant strain JW0940. |
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− | <br> | + | <br>Double digestion was done to confirm the construction.<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/2/20/T--NCKU_Tainan--composite_pOmpA_ompA_oprF.jpg" style="width:35%;"> | <img src="https://2021.igem.org/wiki/images/2/20/T--NCKU_Tainan--composite_pOmpA_ompA_oprF.jpg" style="width:35%;"> | ||
</div> | </div> | ||
− | <p align="center"> | + | <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 | + | <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%;"> | ||
+ | </div> | ||
+ | <p align="center">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)</p> | ||
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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=== |
Latest revision as of 01:40, 22 October 2021
PompA-OmpA/OprF
Description
PompA-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 PompA-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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1142
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]