Difference between revisions of "Part:BBa K2539300"
 
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<partinfo>BBa_K2539300 short</partinfo>
 
<partinfo>BBa_K2539300 short</partinfo>
  
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This construct was built to overexpress alcR, a regulatory protein necessary for the activation of palcA (BBa_K2092002). We acquired all parts from the iGEM distribution kit: a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, strong RBS (BBa_B0034), alcR (BBa_K2092001), and a double terminator (BBa_B0015) to end transcription. PCR checks and sequencing results from Tri-I Biotech confirmed that our final alcR construct is correct.
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AlcR is a gene found in <i>Aspergillus nidulans</i> which encodes for a regulatory protein (Panozzo <i>et al.</i>, 1997). AlcR can bind to the alcA and aldA promoters (PalcA and PaldA, respectively). In the presence both AlcR and ethanol or threonine, genes downstream of these promoters are expressed (Felenbok <i>et al.,</i> 1988).
  
  
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<b><font size="+1">Construct Design</font></b>
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https://static.igem.org/mediawiki/parts/f/fb/T--TAS_Taipei--300construct.jpg
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This construct constitutively express alcR, a regulatory protein necessary for the activation of PalcA. We use a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the alcR sequence (BBa_K2092001), and a double terminator (BBa_B0015) to end transcription.
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<b><font size="+1">PCR Check Results</font></b>
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The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.
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https://static.igem.org/mediawiki/parts/a/a2/T--TAS_Taipei--300pcr.jpg
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<b>PCR check for BBa_K2539300 (A) using VF2 and VR primers. Using these primers, PCR produced a band around the expected size of 3.1 kb.</b>
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<b><font size="+1">References</font></b>
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Felenbok B, Sequeval D, Mathieu M, Sibley S, Gwynne DI, Davies RW. (1988). The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR. Gene, 73(2), 385–396.
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Panozzo C, Capuano V, Fillinger S, Felenbok B. (1997). The zinc binuclear cluster activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. J Biol Chem. 5;272(36):22859-65.
  
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===Characterization===
 
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AlcR is a transcription factor that, along with substances like ethanol, activates palcA. As part of a functional test testing the efficiency of palcA, this construct was ran as a negative control. 
 
  
  

Latest revision as of 12:45, 17 October 2018


AlcR Expression Construct

AlcR is a gene found in Aspergillus nidulans which encodes for a regulatory protein (Panozzo et al., 1997). AlcR can bind to the alcA and aldA promoters (PalcA and PaldA, respectively). In the presence both AlcR and ethanol or threonine, genes downstream of these promoters are expressed (Felenbok et al., 1988).


Construct Design

T--TAS_Taipei--300construct.jpg

This construct constitutively express alcR, a regulatory protein necessary for the activation of PalcA. We use a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the alcR sequence (BBa_K2092001), and a double terminator (BBa_B0015) to end transcription.


PCR Check Results

The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.

T--TAS_Taipei--300pcr.jpg


PCR check for BBa_K2539300 (A) using VF2 and VR primers. Using these primers, PCR produced a band around the expected size of 3.1 kb.


References

Felenbok B, Sequeval D, Mathieu M, Sibley S, Gwynne DI, Davies RW. (1988). The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR. Gene, 73(2), 385–396.

Panozzo C, Capuano V, Fillinger S, Felenbok B. (1997). The zinc binuclear cluster activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. J Biol Chem. 5;272(36):22859-65.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2611
    Illegal BamHI site found at 1615
    Illegal BamHI site found at 2353
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1319
    Illegal AgeI site found at 1447
    Illegal AgeI site found at 1978
  • 1000
    COMPATIBLE WITH RFC[1000]