Team:Paris Bettencourt/Project/Eliminate Smell

Figure 1. Enzymes responsible for body odor in the human axilla (Tauch, 2013).


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).

[figure 3 GC analysis] [Figure 4: phylogenetic tree] [Figure 5: smell test data??]

[Figure 6: megane's stuff??]

1. The Microbiome: Looking for Genes Responsible for Body Odor

Achievements

- Clone AgaA biobrick in E. coli.
- Find Corynebacterium species in axillary samples.
- Find genes related to odor in skin sweat samples.

Introduction

In previous literature, it has been determined that there are a few key enzymes responsible for body odor, such as AgaA, AecD, Ldh, and AckA. Fig. 1 shows a description of the enzymes studied in our project and the reactions corresponding to each enzyme.

The AgaA enzyme in Corynebacterium striatum is found to be a major source of "pungent" or "musky" odor in humans (Kligman, 1981). A specific bacterial aminoacylase cleaves odorant precursors secreted in the human axilla (Acuna,2003), and 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.

Results

In order to analyze the smell created by AgaA, agaA was successfully cloned into E. coli. A noticeable odor was produced by agaA-expressing E. coli grown in selective LB, described 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 test (Fig. 2) as well as analysis by gas chromatography (Fig. 3).

We analyzed human sweat samples for two things. First, we conducted 16S sequencing of the samples collected in order to determine the types of Corynebacterium species present in the sample. The reason we were interested in Corynebacterium was because it is known from literature that one of the main enzyme responsible for the body odor smell (AgaA) is found in Corynebacterium species. Not only did we do 16S sequencing on these samples, but also conducted smell tests on the same samples in order to see if there was a correlation between odor smell and presence of Corynebacterium species.

Second, we conducted Sanger sequencing of axillary sweat samples for the genes in Fig. 1. [MEGANE ADD STUFF HERE ABOUT WHAT YOU DID EXACTLY / HOPED TO DO].

Methods

Cloning AgaA into E. coli

The AgaA enzyme from Corynebacterium (Genbank: AF534871.1) was codon optimized using IDT online tool (Integrated DNA Technologies, Belgium) for E. coli K12 and was commercially synthesized by IDT. PCR was performed to obtain the AgaA construct using a forward primer that contained the Biobrick prefix and a RBS designed using the Salis online tool; and a reverse primer that contained the BioBrick suffix. The PCR product was purified.

The agaA construct and PSB1C3 vector were digested using EcoRI and PstI. After, the agaA construct was purified using the PCR Purification Kit . The vector was gel extracted and purified. Both parts were ligated using Ligase T4.

Competent E. coli Neb Turbo were transformed with the ligation product by heat shock. Then, they were recovered during 2 hours in 200 uL of LB medium at 37ºC and plated in selective plates with LB and chloramphenicol in a concentration of 1 uL/mL. The transformation was confirmed by analytical digestion.

2) CRISPRs: finding natural odorless mutants

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3) Probiotic cream: a cure for body odor

(Figure for the cream.)