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Team:Paris Bettencourt/Project/Odor Library
From 2014.igem.org
Stop And Smell The Roses
Synthetic enzymes can produce odors that humans experience directly, without special instruments. The banana and wintergreeen smell BioBricks are iGEM icons, and a favorite way to introduce genetic engineering. An expanded library of easy-to-use odor enzymes would take synthetic biology to new audiences for creativity, beauty and fun! |
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| Introduction | Results | Methods |
Introduction
Detection of chemical signals from the environment is an intrinsic property of all living organisms (Bargmann 2006). Olfaction is a highly complex phenomena where relations among the stereochemistry of volatile compounds, their ratio within a particular mix, the amount of active olfactory receptors expressed in the smeller, as well as the distribution, and interaction of the different olfactory receptor neurons (ORNs) sum up to generate what we perceive as odor (Buck & Axel 1991; Buck 2004; Lundström & Olsson 2013).
Odors spark neurochemical signals that are processed in different areas of the brain; they trigger complex cognitive processes that affect emotional responses such as motivation and memory (Lenochová et al. 2012). Interestingly, environmental variables such as visual cues and individual variables such as context and diet play a role in odor perception (Gottfried & Dolan 2003; Kuang & Zhang 2014).
Although the precise molecular mechanisms behind odor perception have not been fully understood (Brookes et al. 2007), there has been significant advance in the biosynthesis and applications of organic volatile compounds using bacterial and fungal systems (Korpi et al. 1998; Weisskopf 2013; Callewaert et al. 2014).
Escherichia coli naturally produces indole, an aromatic compound that contributes to the aroma of stool. An E. coli mutant with a deletion of the tnaA gene will not express tryptophanase (TnaA) and therefore won’t produce indole (Li & Young 2013). This mutant can be found in the Keio collection of single-gene knockout mutants (Baba et al. 2006) and was used as the odorless E. coli chassis for the “Smell the roses” library.
Results
We created BioBricks coding for sequences of different enzymes to explore each of the odors described in the palette. These sequences are codon-optmized for produced expression in E. coli and produce the smells that compose the main odor categories perceived by humans.
Methods
Odorless E. coli:
The tnaA deletion mutant E. coli was taken from location 63:E:9 in the Keio collection. Standard microbiology techniques were used to culture it in liquid and solid media. Subsequently, a single colony was taken to make a batch of Ca2Cl chemically competent cells using.
BioBricks:
The coding sequences for BMST1,ATF1, LIMS1 and GGS were extracted from the Registry. The ATF1 and LIMS1 generator BioBricks were taken from previous iGEM distribution kits and were cloned into the pSB1C3 vector downstream of BBa_J23108 constitutive promoter. The BMST1, JMT, and GDS sequences were codon optimized for expression in E. coli. A synthetic ribosome-binding site that includes XmaI and AgeI flanking restriction sites was designed using the RBS calculator from the Salis’ lab. All BioBricks were assembled using standard molecular biology cloning techniques and 3A iGEM assembly.
Characterization:
Transformed E. coli was grown in M9 minimal media with 2% glucose and complete amino acid mix overnight with the appropriate substrate.
| System | Enzyme | Substrate | Concentration | Product |
|---|---|---|---|---|
| Jasmine | JMT | Jasmonic acid | 1 mM | Methyl jasmonate |
| Mint | BMST1 | Salicylic acid | 2 mM | Methyl salicylate |
| Banana | ATF1 | Isoamyl alcohol | 5 mM | Isoamyl acetate |
| Limonene | LIMS1 | Farnesyl diphosphate | Natural | (+)-Limonene |
| Rain | G/G D-synthase | Farnesyl diphosphate | Natural | Geosmin |
| Flowers | BMST1 | Benzoic acid | 5 mM | Methyl benzoate |
| Butter | AldB | Acetolactic acid | - | Acetoin |
| Sweat | agaA | 3-M-2-hexenoic acid | - | 3-HO-3-Methylhexanoic acid |
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