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Team:Paris Bettencourt/Project/TMAU
From 2014.igem.org
Something Fishy
| BACKGROUND Trimethylamine (TMA) is produced by bacteria in the human gut and degraded in the liver by a flavin-containing monooxygenase 3 (FMO3). Trimethylaminuria, or Fish Odor Syndrome, is a rare genetic disease caused by inactivating mutations in the FMO3 gene. Consequently, TMA accumulates in sweat, saliva, and urine, causing a strong fish odor. Patients suffer no other serious symptoms, except a difficult social condition. |
AIMS TMA is also processed by the trimethylamine monooxygenase (Tmm) of Ruegeria pomeroyi, an enzyme similar to human FMO3. If we express this enzyme in human skin bacteria, it should remove TMM from sweat and reduce its unpleasant odor. TMM-expressing bacteria in a cream or spray might be used by trimethylaminuria patients as a stable, inexpensive treatment. |
RESULTS
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| Aims and Achievement | Introduction | Results | Methods | References |
Aims and Achievement
Ruegeria pomeroyi, a bacteria member of the Rhodobacteraceae genus, produces an enzyme called trimethylamine monooxygenase thanks to the tmm (trimethylamine mono-oxygenase) gene. As FMO3, this enzyme degrades trimethylamine into trimethylamine-N-oxide but is adapted to a bacterial expression. The project aims at cloning tmm into E.coli and then into Corynebacterium striatum, one of the most common bacteria of the skin. The new strain would be integrated to the skin microbiome and would suppress the fish odor.
Introduction
Trimethylamine (TMA) is produced in the intestine by Desulfovibrio desulfuricans by fermentation of choline. In healthy patients, the fmo gene allows the degradation of TMA in the liver into a non-volatile compound, TMA oxide. But a mutation in the fmo3 sequence is most of the time the cause of trimethylaminuria: TMA is not degraded and is then excreted in sweat, saliva and urine leading to a strong fish odor. The patients are otherwise healthy but the disease affect their social relationships and can lead to depression. There is currently no cure for this metabolic disorder. Some treatments, often focused on restricting diet, tend to lower the symptoms.
Results
After cloning tmm into a Biobrick vector (pSB1C3), the construct was successfully expressed in E. coli. TMM activity was found in TMM-expressing E. coli but not in empty vector-expressing E. coli. TMM does not only degrade trimethylamine into trimethylamine-N-oxide, but also converts indole into indigo. To measure the activity of TMM, the growth medium was supplemented with tryptophan, a precursor of indole, which is the substrate of TMM. After 14h of culture, cells were pelleted, washed twice with sterile water, resuspended in DMSO and sonicated. TMM activity was determined by measuring the absorbance spectrum of bacterial extractions. Peaks at 620 nm were found in TMM-expressing E.coli cultures supplemented with tryptophan, which was identified as indigo according to absorbance spectrum analysis. Gas chromatography-mass spectrometry (GC/MS) confirmed the activity of TMM by showing a decrease of the concentration of TMA by TMM-expressing E.coli.
Methods
Cloning The tmm gene was produced by gene synthesis (IDT). The final construct was codon-optimized for E. coli expresion and included a strong constitutive promoter and ribosome binding site. For E. coli expression, we cloned the Tmm construct into the standard high-copy BioBrick vector pSB1C3 using XbaI and PstI restriction sites. For expression in C. striatum we used the vector pSEVA351, a universal vector with a high-copy replication origin (Reference)using XbaI and HindIII restriction sites. Indigo absorbance TMM does not only degrade trimethylamine into trimethylamine-N-oxide, but also converts indole into indigo. Measurement of TMM activity was performed on TMM-expressing E.coli using the isolation of indigo protocol inspired of Choi H.S., Kim J.K et al. (2003). Gas chromatography-mass spectrometry
References
- ref1- ref2
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