Difference between revisions of "Part:BBa K3784003"

 
 
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fldH is from the fld gene cluster of Clostridium sporogenes, involved in the conversion of tryptophan to 3-Indolepropionic acid, which express phenyllactate dehydrogenase.
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The fldH 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, fldH encodes phenyllactate dehydratase, which catalyze indolepyruvate to produce indole lactic acid. This enzyme is also the rate-limiting enzyme of the entire reaction
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===Characterization===
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We successfully inserted the fldH gene into the His-6p-MBP-RSFD plasmid and transformed it 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 fldH 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-fldH.png]]
  
 
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Latest revision as of 10:15, 17 October 2021


fldH (Encoding phenyllactate dehydrogenase)

The fldH 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, fldH encodes phenyllactate dehydratase, which catalyze indolepyruvate to produce indole lactic acid. This enzyme is also the rate-limiting enzyme of the entire reaction

Characterization

We successfully inserted the fldH gene into the His-6p-MBP-RSFD plasmid and transformed it 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 fldH is well expressed and the expression levels of internal reference genes in the experimental group (rsmAE) and the control group (rsmAC) were consistent.

BNUZ-fldH.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 997
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]