Difference between revisions of "Part:BBa K3771015"
 
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<partinfo>BBa_K3771015 short</partinfo>
 
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<br><b style="font-size:1.3rem">Description</b>
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<br><b style="font-size:1.3rem">Description</b><br>
  
 
   <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>
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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>
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  <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>
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<br><b style="font-size:1.3rem">Characterization</b><br> 
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<p align="center">Fig. 1. PCR confirmation of <i>P<sub>lacI</sub>-ompA/oprF</i></p>
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<br>Western blot was performed to confirm the expression of OmpA/OprF
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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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1039
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]