Difference between revisions of "Part:BBa K1155000"

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<partinfo>BBa_K1155000 short</partinfo>
 
<partinfo>BBa_K1155000 short</partinfo>
  
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promoter region containing an operator and a promoter controlled by FNR that allow the repression of the downstream gene in anaerobic conditions
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Promoter region containing an operator and a promoter controlled by FNR that allow the repression of the downstream gene in anaerobic conditions
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In ''Escherichia coli'', the switch between aerobic and anaerobic metabolism is partly regulated the transcriptional regulator FNR. This protein has a dual function: it activates genes involved in anaerobic metabolism and represses genes involved in aerobic metabolism. FNR expression is constitutive, but its activity is directly affected by the presence of oxygen which oxidizes of an essential [4Fe-4S] cluster.
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The expression of the ndh gene, encoding an non-proton-translocating NADH dehydrogenase is repressed by FNR in anaerobic conditions. FNR binds at two sites in the promoter region of ndh: a high affinity site (FNRI), and a low affinity, imperfect site (FNRII). In 1997, Meng and colleagues designed a modified ndh promoter region, called Pndh*, with a FNRII site engineered to have a perfect FNR binding site, improving 10 fold the degree of anaerobic repression ''in vivo''.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1155000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1155000 SequenceAndFeatures</partinfo>
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==='''Characterization'''===
 
==='''Characterization'''===
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To characterize BBa_K1155000, we introduced the reporter composite biobrick BBa_K1155003 (based on the blue chromoprotein AmilCP) downstream of BBa_K1155000, using the 3A assembly protocol. We then introduced the resulting plasmid in ''E. coli'' DH5α and cultivated the strain in anaerobic and aerobic conditions in liquid LB medium overnight at 37°C. After centrifugation of the cultures, the cellular pellet had a clear blue/purple color for cells grown in aerobic conditions, whereas the cells grown in anaerobic conditions remained white.
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[[File:PsPfnr3008.jpg|right|400px|caption]]
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'''Tubes 1 and 2''': Cellular pellet of cultures grown in aerobic conditions
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'''Tubes 3 and 4''': Cellular pellet of cultures grown in anaerobic conditions
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==='''Reference'''===
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Meng W., Green J. and Guest J. R. (1997). FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites, Microbiology, 143, 1521-1532.
  
  

Latest revision as of 01:38, 5 October 2013

Pndh*

Promoter region containing an operator and a promoter controlled by FNR that allow the repression of the downstream gene in anaerobic conditions


In Escherichia coli, the switch between aerobic and anaerobic metabolism is partly regulated the transcriptional regulator FNR. This protein has a dual function: it activates genes involved in anaerobic metabolism and represses genes involved in aerobic metabolism. FNR expression is constitutive, but its activity is directly affected by the presence of oxygen which oxidizes of an essential [4Fe-4S] cluster. The expression of the ndh gene, encoding an non-proton-translocating NADH dehydrogenase is repressed by FNR in anaerobic conditions. FNR binds at two sites in the promoter region of ndh: a high affinity site (FNRI), and a low affinity, imperfect site (FNRII). In 1997, Meng and colleagues designed a modified ndh promoter region, called Pndh*, with a FNRII site engineered to have a perfect FNR binding site, improving 10 fold the degree of anaerobic repression in vivo.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

To characterize BBa_K1155000, we introduced the reporter composite biobrick BBa_K1155003 (based on the blue chromoprotein AmilCP) downstream of BBa_K1155000, using the 3A assembly protocol. We then introduced the resulting plasmid in E. coli DH5α and cultivated the strain in anaerobic and aerobic conditions in liquid LB medium overnight at 37°C. After centrifugation of the cultures, the cellular pellet had a clear blue/purple color for cells grown in aerobic conditions, whereas the cells grown in anaerobic conditions remained white.


caption


Tubes 1 and 2: Cellular pellet of cultures grown in aerobic conditions


Tubes 3 and 4: Cellular pellet of cultures grown in anaerobic conditions











Reference

Meng W., Green J. and Guest J. R. (1997). FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites, Microbiology, 143, 1521-1532.