Difference between revisions of "Part:BBa K3771015"
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| − | <br><b style="font-size:1.3rem">Description</b> | + | <br><b style="font-size:1.3rem">Description</b><br> |
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<br><i>P<sub>lacI</sub>-ompA/oprF</i> is part of the IFN-γ sensing system. <i>lacI</i> promoter regulates the constitutive expression of OmpA/OprF. | <br><i>P<sub>lacI</sub>-ompA/oprF</i> is part of the IFN-γ sensing system. <i>lacI</i> promoter regulates the constitutive expression of OmpA/OprF. | ||
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| − | <br><b style="font-size:1.3rem">Biology</b> | + | <br><b style="font-size:1.3rem">Biology</b><br> |
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<br>When there is no <i>lac</i> repressor, the <i>lacI</i> promoter facilitates the constitutive expression of OmpA/OprF. 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>When there is no <i>lac</i> repressor, the <i>lacI</i> promoter facilitates the constitutive expression of OmpA/OprF. 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. | ||
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| − | <br><b style="font-size:1.3rem">Usage</b> | + | <br><b style="font-size:1.3rem">Usage</b><br> |
<br>We ligased <i>ompA/oprF</i> gene and the <i>P<sub>lacI</sub></i> expression vector pSU 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.<br> | <br>We ligased <i>ompA/oprF</i> gene and the <i>P<sub>lacI</sub></i> expression vector pSU 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.<br> | ||
| − | <br><b style="font-size:1.3rem">Characterization</b> | + | <br><b style="font-size:1.3rem">Characterization</b><br> |
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<img src="https://2021.igem.org/wiki/images/e/e6/T--NCKU_Tainan--composite_pLacI_ompA_oprF_.jpg | <img src="https://2021.igem.org/wiki/images/e/e6/T--NCKU_Tainan--composite_pLacI_ompA_oprF_.jpg | ||
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| − | <p align="center">PCR</p> | + | <p align="center">Fig. 1. PCR confirmation of <i>P<sub>lacI</sub>-ompA/oprF</i></p> |
<br>Western blot was performed to confirm the expression of OmpA/OprF | <br>Western blot was performed to confirm the expression of OmpA/OprF | ||
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| + | <img src="https://2021.igem.org/wiki/images/9/9b/T--NCKU_Tainan--results_ompA_wb.jpg" style="width:50%;"> | ||
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| + | <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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Latest revision as of 19:43, 19 October 2021
PlacI-OmpA/OprF
Description
PlacI-ompA/oprF is part of the IFN-γ sensing system. lacI promoter regulates the constitutive expression of OmpA/OprF.
Biology
When there is no lac repressor, the lacI promoter facilitates the constitutive expression of OmpA/OprF. 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
We ligased ompA/oprF gene and the PlacI expression vector pSU 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
Fig. 1. PCR confirmation of PlacI-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 1039
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]