Composite

Part:BBa_K2570009

Designed by: Luying He   Group: iGEM18_FJNU-China   (2018-09-30)


Degrading enzyme complex：araC＋atoD＋atoA＋adhE2

This year, our team aims to tackle environmental problems in daily lives. We focused on harmful bacteria and unpleasant smell under the armpits. From the literature of Andreas Natsch, we knew about that the odorant acid E-3-methyl-2-hexenoicacid (E-3M2H) is abundant and quantitatively dominant human odorant. [1] Therefore, we tried to eliminate such unpleasant axilla odors in an innovative method by degrading the odorant acid. Referring to potential production platform of n-butanol in E. coli [2], we designed a combined part to degrade E-3-methyl-2-hexenoicacid (E-3M2H).

This is a combined part of BBa_K2570006, BBa_K2570007 and BBa_K2570008 degrading main unpleasant axilla odors E-3M2H(E-3-methyl-3-hexenoic acid). This gene cluster expresses three enzymes, of which atoD and atoA from E.coli and adhE2 from Clostridium acetobutylicum ATCC 824. The function of atoDA mediates reductive conversion of 3-methyl 2-hexenoic acid to 3-methyl-2-acetyl-CoA with acetyl-CoA as the CoA donor, for fatty acid activation and its subsequent degradation. [2] AdhE2 catalyzes reduction of 3-methyl-2-acetyl-CoA to 3-methyl 2-hexenol at the expense of NADH. [3]

Fig.1 Possible pathway of degrading the odorant acid E-3M2H.

In our design, the cluster includes an arabinose promoter, strong RBS, atoDA coding region, and adhE2 coding region. atoDA and adhE2 are expressed under the induction of arabinose to achieve degradation of the odorant acid.

Fig2. Circuit of degrading the odorant acid E-3M2H.

We obtained atoD and atoA from E.coli Bw25113 by PCR. AdhE2 was synthesized from a gene synthesis company and was codon optimized of the sequence. Also we did mutation of restriction sites EcoRⅠand PstⅠto meet the requirements of RFC10. We chose pRB1s as the backbone and successfully construct pRB1s-atoD-atoA-adhE2 by gibson assembly. Then we transformed it into the odorless E. coli tnaA-/- and verified its function.

Fig.1 Schematic map of the recombinant plasmid pRB1s-atoD-atoA-adhE2.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2085
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3478
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2731
    Illegal AgeI site found at 3133
    Illegal AgeI site found at 3570
    Illegal AgeI site found at 4504
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


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