Difference between revisions of "Part:BBa K1403015"
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<partinfo>BBa_K1403015 short</partinfo> | <partinfo>BBa_K1403015 short</partinfo> | ||
| − | 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. | + | 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. |
Trimethylamine + NADPH + H+ + O2 = Trimethylamine-N-oxide + NADP+ + H2O | Trimethylamine + NADPH + H+ + O2 = Trimethylamine-N-oxide + NADP+ + H2O | ||
| − | + | The Biobrick contains: | |
<ul> | <ul> | ||
<li>Constitutive promoter <partinfo>BBa_J23108</partinfo></li> | <li>Constitutive promoter <partinfo>BBa_J23108</partinfo></li> | ||
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<li>Stop codon TGA</li> | <li>Stop codon TGA</li> | ||
</ul> | </ul> | ||
| − | + | <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=== | ||
| − | 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. | + | 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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| − | The TMM enzyme is not specific to TMA as a substrate. | + | 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 | + | We took advantage of this indole production activity to characterize the TMM enzyme. |
After transformation, some dark colonies expressing TMM were observed (Fig. 1). | After transformation, some dark colonies expressing TMM were observed (Fig. 1). | ||
[[File:IndigoColsPB.jpg|center|400px]] | [[File:IndigoColsPB.jpg|center|400px]] | ||
| − | <center>'''Figure 1. Dark colonies from transformation on LB+Chloramphenicol plates | + | <center>'''Figure 1. Dark colonies from transformation on LB+Chloramphenicol plates indicate the expression of TMM'''</center> |
| − | 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). | + | 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]] |
<center>'''Figure 2. Average absorbances of DMSO extractions from bacterial lysates shows TMM activity.'''</center> | <center>'''Figure 2. Average absorbances of DMSO extractions from bacterial lysates shows TMM activity.'''</center> | ||
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| − | GC/MS | + | 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. |
[[File:TMM_GCMS.png|center|800px]] | [[File:TMM_GCMS.png|center|800px]] | ||
<center>'''Figure 3. GC/MS analysis of cultures of E. coli expressing TMM or an empty vector (control).'''</center> | <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:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 438
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 262
Illegal AgeI site found at 485 - 1000COMPATIBLE 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).
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.
