Team:Paris Bettencourt/Project/Eliminate Smell

Figure 1. Enzymes responsible for body odor in the human axilla (Tauch, 2013 et al., 2001).


Figure 2. Smell test. 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 produced from 16S sequencing of Corynebacterium from human axillary samples. Corynebacterium was cultured on blood agar plates and the 16S region of selected colonies was sequenced. Sequencing results are presented here in a phylogenetic tree format. The tree was made by creating a multiple sequence alignment (MSA) using the CLUSTALW algorithm and then creating a neighbor-joining tree using the MSA. The length and order of the branches is not accurate due to the evolutionary closeness of the species sampled.

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

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 et al., 1981). A specific bacterial aminoacylase cleaves odorant precursors secreted in the human axilla (Acuna et al., 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, we cloned the gene 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) and plasmid sequencing.

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. Fig. 4 shows the variety of Corynebacterium that we found in human samples after 16S sequencing. Not only did we did the sequencing, 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 to find hotspots for genes ackA and ldh (responsible for body odor).

Methods

1. Cloning agaA into E. coli

The AgaA enzyme from Corynebacterium (Genbank: AF534871.1) was codon optimized using the 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. Find Corynebacterium in human skin samples.

After collecting human axilary samples, we cultured them in blood agar plates to select for Corynebacterium and performed 16S sequencing. We made a multiple sequence alignment (MSA) using the CLUSTAW algorithm and we created a neighbor-joining tree using the MSA. Finally, we obtained a phylogenetic tree for the Corynebacterium found in our samples (Fig. 4).

Figure 4. Phylogenetic tree produced from 16S sequencing of Corynebacterium from human axillary samples. Corynebacterium was cultured on blood agar plates and the 16S region of selected colonies was sequenced. Sequencing results are presented here in a phylogenetic tree format. The tree was made by creating a multiple sequence alignment (MSA) using the CLUSTALW algorithm and then creating a neighbor-joining tree using the MSA. The length and order of the branches is not accurate due to the evolutionary closeness of the species sampled.

2. CRISPR/Cas system as an innovative tool for selecting natural mutants

Introduction

Body odor compounds can often be traced to specific genes in specific strains of the skin microbiome (Fredrich et al., 2013). For example, the musky odor odor of the armpit is produced largely aminoacylase, AgaA, expressed by some strains of Corynebacterium (Natsch et al., 2003). Removing these genes from the microbiome would eliminate their associated odor.

Classical genetic tools can produce gene deletions. Odor-negative deletion mutants, applied to the skin in large numbers, could replace a wild-type strain by the principle of competitive exclusion. However, this approach faces two difficulties:

1) Gene deletions may reduce fitness, rendering the mutant unable to compete with the WT for a niche.
2) Genetically Modified Organisms (GMOs) face special regulations and limited public acceptance.

We propose an alternative strategy to isolate naturally occurring loss-of-function mutants from a complex microbiome. Natural mutants, if they are isolated from the microbiome itself, are likely to retain high fitness. Public acceptance is higher and biosafety concerns are reduced for non-GMO microbes.

The CRISPR-Cas system has been shown to target DNA sequences for cutting with single-base precision. Here we present a CRISPR-based protocol for isolating pre-existing mutants from a mixed population. Mutants that mismatch a CRISPR target sequence will survive transformation with a CRISPR-expressing plasmid and therefore be enriched in the post-transformation population. In principle, CRISPR-based mutant enrichment does not alter the existing mutant genome and the isolated organisms do not meet the conventional definition of GMOs.

Results

3. Probiotic cream: a cure for body odor

Figure X: Cream with Corynebacterium striatum.


Figure X: Cream with fluorescent E. coli.

Ingredients
- Beeswax (known antibacterial)
- Soy milk (substitute medium for tryptophan soy for C. striatum growth)
- Jojoba oil

Formulation of the cream
- Tested cream formulation with RFP E. coli (Fig. X) and checked for growth.
- Tested cream with Corynebacterium striatum (Fig. X).
- Tested raw soy milk versus soy milk hydrolyzed with lemon juice. No significant difference in growth was found.
- Tested shelf life of soy milk and cream formulation. Unspoiled after two months.