Difference between revisions of "Part:BBa K1403015"
 
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<partinfo>BBa_K1403015 short</partinfo>
 
<partinfo>BBa_K1403015 short</partinfo>
  
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This Biobrick encodes trimethylamine mono-oxygenase (<i>tmm</i>), which transforms trimethylamine into trimethylamin-N-oxide. This enzyme is originally expressed by <i>Ruegeria pomeroyi</i> and is similar to human FMO3.
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Trimethylamine + NADPH + H+ + O2 = Trimethylamine-N-oxide + NADP+ + H2O
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The Biobrick contains:
 
<ul>
 
<ul>
 
<li>Constitutive promoter <partinfo>BBa_J23108</partinfo></li>
 
<li>Constitutive promoter <partinfo>BBa_J23108</partinfo></li>
 
<li>Synthetic RBS</li>
 
<li>Synthetic RBS</li>
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<li><i>tmm</i> gene codon-optimized for <i>E. coli</i> (2 illegal EcoRI restriction sites were removed)
 
<li>Histidine tag</li>
 
<li>Histidine tag</li>
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<li>Stop codon TGA</li>
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</ul>
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<br>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
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Bacterial trimethylamine moonoxygenase is similar to mammalian flavin-containing monooxygenase 3 (FMO3) which oxidizes trimethylamine (fish odor) into non-smelly trimethylamine oxide. It also produces indigo (blue pigment) form tryptophan.
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Bacterial trimethylamine mono-oxygenase is similar to mammalian flavin-containing monooxygenase 3 (FMO3), which oxidizes trimethylamine (fish odor) into non-smelly trimethylamine oxide. It also produces indigo (blue pigment) form tryptophan.
  
 
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<partinfo>BBa_K1403015 parameters</partinfo>
 
<partinfo>BBa_K1403015 parameters</partinfo>
 
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===Characterisation===
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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.
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We took advantage of this indole production activity to characterize the TMM enzyme.
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After transformation, some dark colonies expressing TMM were observed (Fig. 1).
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[[File:IndigoColsPB.jpg|center|400px]]
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<center>'''Figure 1. Dark colonies from transformation on LB+Chloramphenicol plates indicate the expression of TMM'''</center>
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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.[[File:Spectrum_indigo.png|center|650px]]
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<center>'''Figure 2. Average absorbances of DMSO extractions from bacterial lysates shows TMM activity.'''</center>
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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.
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[[File:TMM_GCMS.png|center|800px]]
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<center>'''Figure 3. GC/MS analysis of cultures of E. coli expressing TMM or an empty vector (control).'''</center>

Latest revision as of 04:47, 31 October 2014

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).