Microbiology Group: The Search for Anthocyanin Degradation in Nature

Goals

To screen soil samples from around the world for microbes that naturally degrade anthocyanin.
To identify enzymes linked to the most efficient anthocyanin eaters.

BioBricks

BioBrick 1
BioBrick 2
BioBrick 3

Results

Sample origin
Species isolated
How well samples/species degraded quercitin
How well samples/species degraded anthocyanin
Phylogenetic tree of the different species of bacteria and fungus
Common candidate genes

Methods

Microbiome cultivation
Anthocyanin purification
Anthocyanin & Quercitin Measurement
16S rRNA sequencing
Whole genome sequencing
Phylogenetic analysis

Abstract

Standard biochemical activity assays are performed in well-mixed solutions. But enzymes are designed to work at the fabric surface, where real stains are found. To measure our enzyme performance under realistic condtions. Our protocol uses laser-cut fabric circles sized to fit in standard 96-well microplates. In this way, the fabric could be stained, washed, enzyme-treated and incubated using small volume protocols compatible with common lab protocols.
To measure the stain, we used flatbed scanners coupled to custom-made image analysis software. This software is able to identify each circular well and quantify the average pixel color and intensity, which we show is directly proportional to the stain concentration. In this way, we are able to measure the capabilities of our enzymes in conditions that exactly match their real-world application.

Motivation and Background

First challenge: miniature washing machines

A typical garment is composed of several square meters of fabric and a typical washing machine has a volme of 100 liters. While it is possible to perform controlled experiments at this scale, only a few such experiments can be run in parallel in a normal lab. We needed a millimeter-scale system that would allow us to perform hundreds of quantitative assays in parallel.
Experiments on t-shirt scale systems are also complicated by the tendency of liquids to absorb into hydrophilic fabrics like cotton. Small liquid volumes containing stains, detergents or enzymes are quickly wicked into the fabrics and spread over a large area via capillary action. This makes liquids difficult to recover and encourages cross-contamination between different regions of a fabric. Therefore, it was important that each fabric sample in our system be physically enclosed in a micro-well. This allows solutions to be added and removed to a samples under controlled conditions during the course of an experiment.

Second challenge: measuring stain level

Many conventional biochemistry assays are done in well mixed liquid solutions, taking advantage of the uniform absorbance and fluorescence properties to perform quantitative measurements. This is not possible on fabric samples, which are uneven and opaque. We knew that the human eye was able to detect and quantify stains. Therefore, we turned to photographic image analysis software to quantify the color intensity of a 2-D image.

Figure X: Structure and absorbance of malvidin, an anthocyanidin, and quercetin, a flavonol All flavonoids are structured as two phenyl rings and a heterocyclic ring. Anthocyanin itself is structured as a chromane ring with an aromatic ring on C2. Cyanindin and malvidin comprise 90% of the anthocyanins found in nature. These chemicals differ only in their cyclic B groups, and the chromane ring is well conserved in most flavonoids. Therefore, we theorized that the chromane ring itself presented an ideal target for degradation. Based on these criteria, we chose the flavanol quercetin as our anthocyanin substitute. This molecule differs from anthocyanins only in the presence of a carbonyl group (in4?). Additionally, quercetin is present in wine, and contributes to its color. Thus, even in the case where enzymes are isolated that break down quercetin and not anthocyanin, the possibility exists of reducing the color or intensity of wine stains. Finally, co-pigmentation chemical interactions occur between anthocyanin and quercetin, leading to the possibility that quercetin degradation could impact anthocyanin stability (need more background here?).

Results

Collection of the soil samples

Soil samples were collected from France, Spain, Croatia, Namibia and Australia. Samples from the Paris region were collected by members of our team. Other samples were sent by friends, family members, and collaborating iGEM teams. Soil samples were declared to French customs authorities with a Facture Proforma, printed out by the sample donor and included in the shipment.
Upon arrival, samples were washed gently with phosphate-buffered saline (PBS) solution, then left to stand, allowing large particles to settle. The resulting eluate was diluted further with PBS then used directly as a source of soil microbes.

Country	Location	Collector
Paris, France	Clos Monmartre Vineyard	Our Team
Somewhere else	Place name	iGEM friend name

Preparation of the microbe library

For safety, microbial isolation was performed in a fume hood in a BSL 2 facility. More safety information in provided on our safety page.
Microbes were isolated from soil samples using either selective or nonselective plating. In nonselective plating, soil eluate was serially diluted and plated on rich media (Trypic Soy Agar, TSA) or minimal media (M9 Glucose). In selective plating, soil eluate was used to innoculate a 20 mL culture of M9 quercitin media, in which quercitin was the only supplied carbon source. The resulting culture was incubated at 37 C for 48 hours, then serially diluted and plated on TSA. The purpose of selective plating was to enrich for microbes with the ability to metabolize quercitin.
In each case, single colonies were isolated then re-streaked to eliminate potential contamination. To maximize the library diversity, we preferentially chose colonies with unique morphology and we took no more than 5 clones from a single soil sample. After isolation, we performed colony PCR with universal 16s rRNA primers (see methods). Sequencing the resulting PCR products allowed us to identify the strains and position them within the greater bacterial taxonomy.

Quanitification of quercitin degradation

Single colonies from the selective and nonselective microbe libraries were innoculated in M9 Glucose media containing X g of quercitin. Samples were cultured at 37 C for six days. Following incubaction, samples were centrifuged to remove cells and contaminants. Quercitin was measured by absorbance at 375 nm. Of 182 strains tested, 50 produced quercitin levels significantly lower than controls (Figure X). 20 strain produced more than 50% quercitin degradation and 2 strains degraded quercitin more than 80%.
Both selective an nonselective plating methods were able to produce quercitin-degrading strains. 4 of the 5 strains showing the most quercitin degradation were obtained by selective plating. However, 40 of the 50 strains showing significant degradation were obtained by nonselective plating.

Phylogenetic analysis of quercitin degradation

Testing microbes with real wine and real fabrics

Mining genomes for quercitin-degrading enzymes

Figure X: Quercitin degradation by 189 microbes collected from global soil samples Single colonies were inoculated in M9 minimal medium and grown for 6 days. Quercitin was measured by absorbance.

Figure X: Quercitin degradation detail The top-performing strains included isolated from both selective and nonselective media. Strains marked with an asterisk were selected for further investigation.

Methods

Anthocyanin isolation protocol

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Preparation of fabric samples for panning

Fabrics were washed with x prior to panning to remove coatings, preservatives or other treatments that may have been applied in the factory.

Detailed protocol for phage display

10 µl of phage was mixed with 1 gram of fabric...

Sequence similarity was calculated as BLOSUM. Trees were made using Geneious with nearest neighbor joining.

Sequence clustering analysis

Binding quantification with ELISA

Attributions

This project was done mostly by Antoine Villa Antoine Poirot and Sébastien Gaultier. Put here everyone who helped including other iGEM teams

References

Sweetlove, L. J., & Fernie, A. R. (2013). The Spatial Organization of Metabolism Within the Plant Cell. Annual Review of Plant Biology, 64(1), 723–746.
Lee, H., DeLoache, W. C., & Dueber, J. E. (2012). Spatial organization of enzymes for metabolic engineering. Metabolic Engineering, 14(3), 242–251.
Pröschel, M., Detsch, R., Boccaccini, A. R., & Sonnewald, U. (2015). Engineering of Metabolic Pathways by Artificial Enzyme Channels. Frontiers in Bioengineering and Biotechnology, 3(Pt 5), 123–13.