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.

Example figure box

Results

First generation: Wax-based

Second generation: PDMS-based

Second generation: Microplate-based

Methods

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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.