Team:Paris Bettencourt/Project/Eliminate Smell

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BACKGROUND Sweat is initially odor-less, but bacteria in your skin microbiome can process some sulfurous compounds present in sweat to release volatile and odorous compounds. In "Don't Sweat It," we are trying to find natural mutants of the genes that produce odorous compounds, and allow us to smell like ourselves.

AIMS 1. Find the bacteria and genes responsible for body odor in human sweat samples. 2. Develop CRISPRs that target the bacteria responsible for body odor in order to find natural odorless strains. 3. Formulate a probiotic deodorant cream that contains the odorless natural mutants of the bacteria to cure body odor.

RESULTS 1. MICROBIOME RESULTS... Found Corynebacterium species in skin samples..genes?? Made biobrick of agaA (main gene responsible for body odor) in pSB1C3. 2. CRISPR results...in E. coli? 3. Made a DIY formulation of probiotic cream

Smell test:

Figure 2:14 people smelled two tubes of E. coli grown to saturation in LB. One culture carried synthetic agaA and other an empty vector control. 13 people out of 14 rated the E. coli carrying agaA as more smelly (pink) than the control(violet). Experiments were double-blind. Significance was confirmed by Chi square test (p-value = 0.001341).

GC:
We performed a GC analysis in the E. coli expressing agaA and used E. coli transformed with an empty vector as a control. COMPOUND X was detected in the E. coli expressing agaA .

(GC FIGURE)

Corynebacterium striatum was found in natural sweat samples. Via 16s sequencing, we found different Corynebacterium species:

(PHILOGENETIC TREE)

Additionally, we conducted a smell test on these samples.

(FIGURE)

Figure 1:Enzymes responsible for body odor in the human axile (Tauch (2013). Daily battle against body odor: towards the activity of the axillary microbiota. Trends in Microbiology 21(6):305–312).

1) Microbiome study: looking for genes responsible for body odor

There are several enzymes responsible for body odor: agaA, aecD, L-Ldh, D-Ldh, Ldh, and AckA. We Sanger-sequenced human axilary sweat samples for these genes.
(MÉGANE'S FIGURE)
(MÉGANE'S RESULTS: XXX genes are responisble for body odor).

The AgaA enzyme of Corynebacterium striatum is a major source of "pungent" or "musky" aromatics in human body odor (Acuna G. (2003). A specific bacterial aminoacylase cleaves odorant precursors secreted in the human axilla. J Biol Chem. 278 (8), 5718-27). It hydrolyzes 3-methyl-2-hexenoyl-glutamine (3M2H-gln) into 3-methyl-2-hexenoic acid (3M2H) and free glutamine. The enzyme is known to have a low specificity for the acyl group, and to act on a range of glutamine conjugates. We cloned agaA into the standard BioBrick vector, and expressed it in E. coli.

agaA was successfully cloned into E. coli.

A noticeable odor was produced by agaA-expressing E. coli grown in selective LB, described variously as "beer-like" or "cheese-like". We took this to be evidence that the enzyme was functional and acting on a non-native substrate in LB media. We confirmed this observation with a formal smell text (Figure 3) and GC analysis (Figure 4).

2) CRISPRs: finding natural odorless mutants

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3) Probiotic cream: a cure for body odor

(Figure for the cream.)

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