Difference between revisions of "Part:BBa K2092009"

 
 
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<partinfo>BBa_K2092009 short</partinfo>
 
<partinfo>BBa_K2092009 short</partinfo>
  
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PalcA is one of the strongest inducible promoters in Aspergillus nidulans commonly used to overexpress proteins [1]. It has been shown that PalcA promoter is also functional in monocotyledonous plant sugar cane [2] and Escherichia coli (E.coli) [3]. Its transcriptional activation is dependent on the binding of its positive transcriptional regulator AlcR with various substrates that employ a hydroxyl group, for example ethanol and threonine.
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The alcA promoter, P<i>alcA</i>, is originally found in <i>Aspergillus nidulans</i> as a part of the ethanol regulon.  P<i>alcA</i> requires its positive transcriptional regulator AlcR to regulate the expression of gene <i>alcA</i>. Gene <i>alcA</i> encodes for alcohol dehydrogenase I (ADHI) which facilitates the interconversion between alcohols and carbonyls.  This part consists of a P<i>alcA</i> (BBa_K2092002, https://parts.igem.org/Part:BBa_K2092002) variant tagged with a blue chromoprotein, which makes it capable to produce blue colour upon the binding of its ethanol-activated transcription factor AlcR.
  
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The native PalcA consists of 3 AlcR binding sites. The number and position of the AlcR binding sites on the PalcA are crucial in determining its transcriptional activation strength. It has also been shown that each AlcR target in the PalcA contributes differently to the activation of the downstream protein expression [1]. In contrast to the native construct (three AlcR binding sites abc), this variant consists only two binding sites bc. Previous study on sugarcane has shown that there is a 70% decrease in promoter strength for PalcA with only binding sites bc [2].
 
  
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This part allows detection of ethanol by coupling it with another composite part, <a href="https://parts.igem.org/Part:BBa_K2092008"> BBa_K2092008</a>, which constitutively expresses its transcription factor, AlcR.  Blue color development can be seen in presence of ethanol.
 
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===Usage and Biology===
 
===Usage and Biology===
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P<i>alcA</i> is one of the strongest inducible promoters in <i>Aspergillus nidulans</i> commonly used to overexpress proteins [1].  It has been shown that P<i>alcA</i> promoter is also functional in monocotyledonous plant sugar cane [2] and <i>Escherichia coli (E.coli)</i> [3]. Its transcriptional activation is dependent on the binding of its positive transcriptional regulator AlcR with various substrates that employ a hydroxyl group, for example ethanol and threonine. 
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The native P<i>alcA</i> consists of 3 AlcR binding sites.  The number and position of the AlcR binding sites on the P<i>alcA</i> are crucial in determining its transcriptional activation strength.  It has also been shown that each AlcR target in the P<i>alcA</i> contributes differently to the activation of the downstream protein expression [1].  In contrast to the native construct (three AlcR binding sites <i>abc</i>), this variant consists only two binding sites <i>bc</i>. Previous study on sugarcane has shown that there is a 70% decrease in promoter strength for P<i>alcA</i> with only binding sites <i>bc</i> [2]. 
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This part allows detection of ethanol by coupling it with another composite part, BBa_K2092008 (https://parts.igem.org/Part:BBa_K2092008), which constitutively expresses its transcription factor, AlcR.  Blue colour development can be seen in presence of ethanol.
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===References===
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[1] Felenbok, B., Sequeval, D., Mathieu, M., Sibley, S., Gwynne, D.I. and Davies, R.W. (1988). The ethanol regulon in Aspergillus nidulans: Characterization and sequence of the positive regulatory gene alcR. <i>Gene</i>, 73(2), 385–396.
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[2] Kinkema, M., Geijskes, R.J., Shand, K., Coleman, H.D., De Lucca, P.C., Palupe, A., Harrison, M.D., Jepson, I., Dale, J.L. and Sainz, M.B. (2013). An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. <i>Plant Molecular Biology</i>, 84(4-5), 443–454.
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[3] Hemmati, H. and Basu, C. (2015). Transcriptional analyses of an ethanol inducible promoter in Escherichia coli and tobacco for production of cellulase and green fluorescent protein. <i>Biotechnology & Biotechnological Equipment</i>, 29(6), 1043–1052.
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Latest revision as of 01:25, 19 October 2016

PalcA(var) + RBS (B0034) +amilCP

The alcA promoter, PalcA, is originally found in Aspergillus nidulans as a part of the ethanol regulon. PalcA requires its positive transcriptional regulator AlcR to regulate the expression of gene alcA. Gene alcA encodes for alcohol dehydrogenase I (ADHI) which facilitates the interconversion between alcohols and carbonyls. This part consists of a PalcA (BBa_K2092002, https://parts.igem.org/Part:BBa_K2092002) variant tagged with a blue chromoprotein, which makes it capable to produce blue colour upon the binding of its ethanol-activated transcription factor AlcR.


Usage and Biology

PalcA is one of the strongest inducible promoters in Aspergillus nidulans commonly used to overexpress proteins [1]. It has been shown that PalcA promoter is also functional in monocotyledonous plant sugar cane [2] and Escherichia coli (E.coli) [3]. Its transcriptional activation is dependent on the binding of its positive transcriptional regulator AlcR with various substrates that employ a hydroxyl group, for example ethanol and threonine.

The native PalcA consists of 3 AlcR binding sites. The number and position of the AlcR binding sites on the PalcA are crucial in determining its transcriptional activation strength. It has also been shown that each AlcR target in the PalcA contributes differently to the activation of the downstream protein expression [1]. In contrast to the native construct (three AlcR binding sites abc), this variant consists only two binding sites bc. Previous study on sugarcane has shown that there is a 70% decrease in promoter strength for PalcA with only binding sites bc [2].

This part allows detection of ethanol by coupling it with another composite part, BBa_K2092008 (https://parts.igem.org/Part:BBa_K2092008), which constitutively expresses its transcription factor, AlcR. Blue colour development can be seen in presence of ethanol.


References

[1] Felenbok, B., Sequeval, D., Mathieu, M., Sibley, S., Gwynne, D.I. and Davies, R.W. (1988). The ethanol regulon in Aspergillus nidulans: Characterization and sequence of the positive regulatory gene alcR. Gene, 73(2), 385–396.

[2] Kinkema, M., Geijskes, R.J., Shand, K., Coleman, H.D., De Lucca, P.C., Palupe, A., Harrison, M.D., Jepson, I., Dale, J.L. and Sainz, M.B. (2013). An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. Plant Molecular Biology, 84(4-5), 443–454.

[3] Hemmati, H. and Basu, C. (2015). Transcriptional analyses of an ethanol inducible promoter in Escherichia coli and tobacco for production of cellulase and green fluorescent protein. Biotechnology & Biotechnological Equipment, 29(6), 1043–1052.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 274
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]