Team:Paris Bettencourt/Project/Eliminate Smell

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)

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

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).

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 Coryneacterium 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).

Motivation

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Aims

Instead of killing these bacteria -such as deodorants do-, we want to find a a natural strain that does not have agaA in it, and create a probiotic cream.

In order to do so, the 'Don't sweat it team' has followed these steps:
1)agaA BioBrick: Create a E. coli strain that contains agaA
2)CRISPRs selection: Select a Corynebacterium strain that has a mutant agaA gene.
3) Natural strains: find a natural strain that contains a mutant agaA or no agaA at all. We will use samples taken from volunteers and perform deep sequencing and do bacteria identification and isolation.
4) Probiotic cream: Design a product that can be used as a probiotic deodorant using a natural Corynebacterium strain that does not release sulphur compounds.

Results

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