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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alcohol dehydrogenase is an enzyme, which catalyzes the reaction of alcohols to their corresponding aldehydes and ketones as well as the reverse reaction. With the help of the PTS1 sequence the enzyme is targeted to the peroxisome allowing to use the ADH for pathways implemented in the peroxisome.
  
 
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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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Latest revision as of 03:52, 2 November 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