Difference between revisions of "Part:BBa K3784000"
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<partinfo>BBa_K3784000 short</partinfo>
 
<partinfo>BBa_K3784000 short</partinfo>
  
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acdA is from the fld gene cluster of Clostridium sporogenes, involved in the conversion of tryptophan to 3-Indolepropionic acid, which encodes acyl-CoA dehydrogenase.
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acdA is from the FLD gene cluster of Clostridium sporogenes, involved in the conversion of tryptophan to 3-Indolepropionic acid. In the five-step metabolic pathway we reconstructed, acdA encodes 3-(aryl)acrylate reductase, which catalyze indole acrylic acid to produce indole propionic acid.
  
 
===Characterization===
 
===Characterization===
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We linked acdA fragment and pET-28a plasmid, then transferred the recombinant plasmid into E. coli and test it by colony PCR.
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We successfully inserted the acdA into the His-6p-MBP-RSFD plasmid, which is transcribed by a self-contained promoter cloned from Clostridium sporogenes.
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[[File:T--bnuz_china--dianyong.jpeg]]
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We transformed the plasmid into E. coli BL21(DE3) and induced it to express. After extracting total RNA, we obtained its cDNA by reverse transcription. We used cDNA as a template to amplify the fragment, then checked it by agarose gel electrophoresis. We chose rsmA (16S rRNA m(6)2A1518, m(6)2A1519 dimethyltransferase) as the internal reference gene and set a negative control. The acdA is well expressed and the expression levels of internal reference genes in the experimental group (rsmAE) and the control group (rsmAC) were consistent.
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[[File:BNUZ-acda.png]]
  
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===Usage and Biology===
 
  
 
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Latest revision as of 08:17, 17 October 2021


acdA (encoding 3-(aryl) acrylate reductase)

acdA is from the FLD gene cluster of Clostridium sporogenes, involved in the conversion of tryptophan to 3-Indolepropionic acid. In the five-step metabolic pathway we reconstructed, acdA encodes 3-(aryl)acrylate reductase, which catalyze indole acrylic acid to produce indole propionic acid.

Characterization

We successfully inserted the acdA into the His-6p-MBP-RSFD plasmid, which is transcribed by a self-contained promoter cloned from Clostridium sporogenes. We transformed the plasmid into E. coli BL21(DE3) and induced it to express. After extracting total RNA, we obtained its cDNA by reverse transcription. We used cDNA as a template to amplify the fragment, then checked it by agarose gel electrophoresis. We chose rsmA (16S rRNA m(6)2A1518, m(6)2A1519 dimethyltransferase) as the internal reference gene and set a negative control. The acdA is well expressed and the expression levels of internal reference genes in the experimental group (rsmAE) and the control group (rsmAC) were consistent.

BNUZ-acda.png


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 291
    Illegal SpeI site found at 619
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 291
    Illegal SpeI site found at 619
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1141
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 291
    Illegal SpeI site found at 619
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 291
    Illegal SpeI site found at 619
  • 1000
    COMPATIBLE WITH RFC[1000]