Difference between revisions of "Part:BBa K2271116"
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The ADH we used for our project is from Pichia pastoris. It converts (+)-trans-nootkatol into (+)-nootkatone by oxidation, using NAD+ as a cofactor. Usually an additionally cofactor regeneration is not necessary, as BM3 degrades NADH+H+ into NAD+ and ADH regenerates it. But because of the improved (+)-nootkatone producing function of BM3, another cofactor regenerating enzyme is necessary. In recent studies it was shown that Glucose Dehydrogenases (GDH) are appropriate enzymes for this attempt.
 
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This part additionally holds a PTS1 sequence for peroxisomal PEX5 import. For cytosolic expression please choose part: BBa_K2271115
 
  
 
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===Usage and Biology===
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<partinfo>BBa_K2271116 parameters</partinfo>
 
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===Usage and Biology===
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We used an alcohol dehydrogenase(ADH) as part of our nootkatone pathway. The ADH we used for our project is from Pichia pastoris.
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It converts (+)-trans-nootkatol into (+)-nootkatone by oxidation, using NAD+ as a cofactor. A cofactor regeneration is not necessary in our case, because BM3, another enzyme of our nootkatone pathway, degrades NADH+H+ into NAD+.
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In nature ADH reduces or oxidizes alcohols to their specific aldehydes or ketones with help of NAD+.
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This part additionally holds a PTS1 sequence for peroxisomal PEX5 import. For cytosolic expression please choose part: BBa_K2271115
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<br>
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==Characterization==
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<div style="text-align:justify;">
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We verified the expression of ADH PTS1 via western blot. Therefore, we have a 3xFLAG-6xHis-Tag as a part in our plasmids, we can use for the antibodies. The estimated atomic mass of ADH PTS1 is 38kDa.
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[[File:T--Cologne-Duesseldorf--western-blot-ADH.png|thumb|none|
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    <strong>Protein abundance in WT and transformed cells from <i>Saccharomyces cerevisiae</i>:</strong> Protein abundance was detected using 6x His Tag Antibody. WT = wild type, ADH = Alcohol Dehydrogenase, PTS1 = Peroxisom Targeting Signal 1]]
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==References==
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<div style="text-align:justify;">

Revision as of 18:53, 30 October 2017

ADH PTS1

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 880
    Illegal BamHI site found at 2047
    Illegal XhoI site found at 2083
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2433
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

We used an alcohol dehydrogenase(ADH) as part of our nootkatone pathway. The ADH we used for our project is from Pichia pastoris. It converts (+)-trans-nootkatol into (+)-nootkatone by oxidation, using NAD+ as a cofactor. A cofactor regeneration is not necessary in our case, because BM3, another enzyme of our nootkatone pathway, degrades NADH+H+ into NAD+. In nature ADH reduces or oxidizes alcohols to their specific aldehydes or ketones with help of NAD+.


This part additionally holds a PTS1 sequence for peroxisomal PEX5 import. For cytosolic expression please choose part: BBa_K2271115



Characterization

We verified the expression of ADH PTS1 via western blot. Therefore, we have a 3xFLAG-6xHis-Tag as a part in our plasmids, we can use for the antibodies. The estimated atomic mass of ADH PTS1 is 38kDa.


Protein abundance in WT and transformed cells from Saccharomyces cerevisiae: Protein abundance was detected using 6x His Tag Antibody. WT = wild type, ADH = Alcohol Dehydrogenase, PTS1 = Peroxisom Targeting Signal 1


References