Generator

Part:BBa_K1403015

Designed by: Clovis & François   Group: iGEM14_aris_Bettencourt   (2014-10-06)

Trimethylamine moonoxygenase (tmm) expression cassette

This Biobrick encodes trimethylamine mono-oxygenase (tmm), which transforms trimethylamine into trimethylamin-N-oxide. This enzyme is originally expressed by Ruegeria pomeroyi and is similar to human FMO3.

Trimethylamine + NADPH + H+ + O2 = Trimethylamine-N-oxide + NADP+ + H2O

The Biobrick contains:

  • Constitutive promoter BBa_J23108
  • Synthetic RBS
  • tmm gene codon-optimized for E. coli (2 illegal EcoRI restriction sites were removed)
  • Histidine tag
  • Stop codon TGA


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 438
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 262
    Illegal AgeI site found at 485
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterisation

The TMM enzyme is not specific to TMA as a substrate. The enzyme is also known to oxidize indole to indoxyl, which dimerizes into the well known blue pigment indigo. Indole is a natural product of tryptophan metabolism in E. coli. We took advantage of this indole production activity to characterize the TMM enzyme. After transformation, some dark colonies expressing TMM were observed (Fig. 1).

IndigoColsPB.jpg
Figure 1. Dark colonies from transformation on LB+Chloramphenicol plates indicate the expression of TMM


E. coli that were cultured in LB supplemented with tryptophan (2 g/L) produced a deep blue pigment with absorbance properties matching those of indigo (Fig. 2). Indigo production was minimal or absent when TMM expression or tryptophan were not present.
Spectrum indigo.png
Figure 2. Average absorbances of DMSO extractions from bacterial lysates shows TMM activity.



GC/MS demonstrated the degredation of trimethylamine, thereby confirming the activity of TMM (Fig. 3). The GC/MS was performed on extractions from cultures of TMM-expressing E. coli (TMM) and on a control expressing an empty vector in a LB medium supplemented with trimethylamine (1 mM). The results show a significant decrease (p-value = 0,0199) in the concentration of TMA in TMM-expressing E.coli (Fig. 3D). These data confirm the efficiency of fish odor degradation by TMM.

TMM GCMS.png
Figure 3. GC/MS analysis of cultures of E. coli expressing TMM or an empty vector (control).
[edit]
Categories
Parameters
None