Part:BBa_K1403015

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

It 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. It 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 advangage of this indole production activity to characterize the TMM enzyme. After transformation, some dark colonies expressing TMM were observed (Fig. 1).

Figure 1. Dark colonies from transformation on LB+Chloramphenicol plates suspect 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 indigo (Fig. 2). Without TMM expression or without tryptophan, indigo production was minimal or absent.

Figure 2. Average absorbances of DMSO extractions from bacterial lysates shows TMM activity.

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

Figure 3. GC/MS analysis of cultures of E. coli expressing TMM or an empty vector (control).