Difference between revisions of "Part:BBa K3771016"
 
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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>
 
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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>
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<br><b style="font-size:1.3rem">Usage</b>
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<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><b style="font-size:1.3rem">Characterization</b>
 
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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>
 
  
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<br>Double digestion was done to confirm the construction.<br>
  
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<img src="https://2021.igem.org/wiki/images/2/20/T--NCKU_Tainan--composite_pOmpA_ompA_oprF.jpg" style="width:35%;">
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<p align="center">Fig. 1. Double digestion check of <i>P<sub>ompA</sub>-ompA/oprF</i></p>
  
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<br>Western blot was performed to confirm the expression of OmpA/OprF.<br>
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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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<!-- Add more about the biology of this part here
 
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===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


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]