Difference between revisions of "Part:BBa K3866015"

 
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<partinfo>BBa_K3866015 short</partinfo>
 
<partinfo>BBa_K3866015 short</partinfo>
  
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<p>The production of butyrate by bacteria can be possible with a set of genes. The superpart 1 includes only four of the nine genes that catalyze the formation of butyric acid. Those are PhaA, PhaB, Crt and Ter genes.
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[[File:T--Thessaly--superpart1.png|700px|thumb|none|<i><b>Fig.2:</b>PhaA, PhaB, Crt, Ter genes constructed in an operon in a PUPD2 vector, with compatible ends for Golden Braid Cloning</i>]]
  
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===Usage and Biology===
 
===Usage and Biology===
  
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First, PhaA and BktB are two β-ketothiolases  that carry out the homo-fusion of two acetyl-CoA moieties into acetoacetyl-CoA. Then, the acetoacetyl-CoA is reduced to (S)-3-hydroxybutyryl-CoA (3-HB-CoA) by NADH-dependent Hbd or (R)-3-HB-CoA by NADPH-dependent PhaB. Both (R)-3-HB-CoA and (S)- 3-HB-CoA can be converted to (R)-3-HB and (S)-3-HB, respectively, by a CoA-removing enzyme, such as Pct, TesB, or Ptb/Buk.  Alternatively, (R)-3-HB-CoA and (S)-3-HB-CoA can be converted to crotonyl-CoA by PhaJ and Crt, respectively. Crotonyl-CoA is then reduced to butyryl-CoA by NADH-dependent Ter, and subsequently converted to butyrate by one of the above CoA-removing enzymes.
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<span class='h3bb'>Sequence and Features</span>
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===Design Notes===
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The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 <bbpart>BBa_K3505007</bbpart> and has overhangs compatible for GoldenBraid cloning. The Promoter has position B3-B5.
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[[File:T--Thessaly--GB-GGAG-AATG.jpeg|700px|thumb|none|<i><b>Fig.1:</b>The overhangs of this part in the GoldenBraid Grammar</i>]]
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===Verification and Cloning===
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[[File:T--Thessaly--superpart1gel.png|350px|thumb|none|<i><b>Fig.2:</b>PhaA,PhaB,Crt,Ter genes into a PUPD2 vector. Digestion with EcoRV
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Expected bands at:
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*4807 bps
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*1262 bps</i>]]
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===Sequence and Features===
 
<partinfo>BBa_K3866015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3866015 SequenceAndFeatures</partinfo>
  
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===Source===
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Synthesized by Twist Biosciences.
  
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===References===
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===Functional Parameters===
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*Miscevic D, Srirangan K, Kefale T, Abedi D, Moo-Young M, Chou CP. Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli. Appl Microbiol Biotechnol. 2019 Jul;103(13):5215-5230. doi: 10.1007/s00253-019-09815-x. Epub 2019 May 2. PMID: 31049621.
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<partinfo>BBa_K3866015 parameters</partinfo>
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Latest revision as of 19:29, 26 September 2021


PhaA-RBS-PhaB-RBS-Crt-RBS-Ter GB compatible with B3-B5

The production of butyrate by bacteria can be possible with a set of genes. The superpart 1 includes only four of the nine genes that catalyze the formation of butyric acid. Those are PhaA, PhaB, Crt and Ter genes.

Fig.2:PhaA, PhaB, Crt, Ter genes constructed in an operon in a PUPD2 vector, with compatible ends for Golden Braid Cloning

Usage and Biology

First, PhaA and BktB are two β-ketothiolases that carry out the homo-fusion of two acetyl-CoA moieties into acetoacetyl-CoA. Then, the acetoacetyl-CoA is reduced to (S)-3-hydroxybutyryl-CoA (3-HB-CoA) by NADH-dependent Hbd or (R)-3-HB-CoA by NADPH-dependent PhaB. Both (R)-3-HB-CoA and (S)- 3-HB-CoA can be converted to (R)-3-HB and (S)-3-HB, respectively, by a CoA-removing enzyme, such as Pct, TesB, or Ptb/Buk. Alternatively, (R)-3-HB-CoA and (S)-3-HB-CoA can be converted to crotonyl-CoA by PhaJ and Crt, respectively. Crotonyl-CoA is then reduced to butyryl-CoA by NADH-dependent Ter, and subsequently converted to butyrate by one of the above CoA-removing enzymes.



Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 BBa_K3505007 and has overhangs compatible for GoldenBraid cloning. The Promoter has position B3-B5.

Fig.1:The overhangs of this part in the GoldenBraid Grammar

Verification and Cloning

Fig.2:PhaA,PhaB,Crt,Ter genes into a PUPD2 vector. Digestion with EcoRV Expected bands at: *4807 bps *1262 bps

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 357
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 357
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3315
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 357
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 357
    Illegal NgoMIV site found at 122
    Illegal NgoMIV site found at 226
  • 1000
    COMPATIBLE WITH RFC[1000]

Source

Synthesized by Twist Biosciences.

References

  • Miscevic D, Srirangan K, Kefale T, Abedi D, Moo-Young M, Chou CP. Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli. Appl Microbiol Biotechnol. 2019 Jul;103(13):5215-5230. doi: 10.1007/s00253-019-09815-x. Epub 2019 May 2. PMID: 31049621.