Difference between revisions of "Part:BBa K1587004"
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===BioBrick Description===
 
===BioBrick Description===
  
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Constitutive promoter p(Bla). Crotonyl-coA reductase ccr coding region. 3-hydroxybutyryl-CoA dehydrogenase hbd coding region. 3-hydroxybutyryl-CoA dehydratase crt coding region. Acyl-CoA thioesterase 2 tesB coding region. acetyl-CoA acetyltransferase atoB coding region. Every genes are framed by strong RBS based on Ron Weiss thesis. This operon ends with an artificial terminator according to <a target="blank" href="https://parts.igem.org/Part:BBa_B1006">BBa_B1006</a>.<br>
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Constitutive promoter p(Bla). Crotonyl-coA reductase ccr coding region. 3-hydroxybutyryl-CoA dehydrogenase hbd coding region. 3-hydroxybutyryl-CoA dehydratase crt coding region. Acyl-CoA thioesterase 2 tesB coding region. acetyl-CoA acetyltransferase atoB coding region. Every genes are framed by strong RBS based on Ron Weiss thesis. This operon ends with an artificial terminator according to[https://parts.igem.org/Part:BBa_B1006 BBa_B1006].<br>
 
Due to a lack of time we decided to have this construction synthesised instead of using the assembly protocol.
 
Due to a lack of time we decided to have this construction synthesised instead of using the assembly protocol.
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===Usage and Biology===
 
===Usage and Biology===

Revision as of 16:38, 18 September 2015

Butyrate synthesis pathway regulated by constitutive promoter p(Bla)

BioBrick Description

Constitutive promoter p(Bla). Crotonyl-coA reductase ccr coding region. 3-hydroxybutyryl-CoA dehydrogenase hbd coding region. 3-hydroxybutyryl-CoA dehydratase crt coding region. Acyl-CoA thioesterase 2 tesB coding region. acetyl-CoA acetyltransferase atoB coding region. Every genes are framed by strong RBS based on Ron Weiss thesis. This operon ends with an artificial terminator according toBBa_B1006.
Due to a lack of time we decided to have this construction synthesised instead of using the assembly protocol.


Usage and Biology

TLSE SHLAGAGA.png
Figure 1: Genetic construct to produce butyrate via glucose


TLSE ButMetabo.jpg
Figure 2: Metabolic pathway to produce butyrate via glucose using BBa_K1587004 in E. coli




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 36
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1717
    Illegal BamHI site found at 3634
    Illegal XhoI site found at 1407
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 332
    Illegal AgeI site found at 1254
    Illegal AgeI site found at 2900
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1752
    Illegal BsaI.rc site found at 2169
    Illegal BsaI.rc site found at 3237
    Illegal SapI site found at 123
    Illegal SapI.rc site found at 3249

Sequencing has been made thanks to 7 primers specifics for these genes:
VF2, CCR, hbd, crt, testB, atoB and VR


Experiments

We used BBa_K1587004 in order to produce butyrate

TLSE Butprod.jpg

Figure 3: NMR analysis of butyrate production in E. coli with BBa_K1587004 and wt


Unfortunatly NMR analysis did not yield a significant over production of butyrate in E. coli. However we observed a statistically significant change in the production of other fermentation products such as ethanol or acetate.