Introduction

The microbiology team aims to identify wine stain degrading microorganisms.

Basing this bioremediation-inspired approach on the two following articles:

• Bioremediation of phenol by alkaliphilic bacteria isolated from alkaline lake of Lonar, India
  P.P. Kanekar, S.S. Sarnaik and A.S. Kelkar. Journal of applied microbiology supplement 1999.
• Bacteria Subsisting on Antibiotics
  Gautam Dantas, Morten O. A. Sommer, Rantimi D. Oluwasegun, George M. Church. Science 2012)

We hypothesize that these microorganisms could be preferably discovered in vineyard soil samples.
Indeed, it is highly probable that a rich-anthocyanin environment such as a vineyard, would host microbes with the desired degradation skills. In addition, the chance to find a microbe able to digest efficiently a wine stain, with its proper anthocyanin composition, in terms of anthocyanin diversity, and abundance, is theoretically increased.
To ensure the widest microorganisms diversity, we used soil samples from all around the world (Australia, Spain, Namibia, France, Croatia), particularly through collaboration with different iGEM teams.

This bacteria identification was based on two different approaches:

• We try to create a bacteria database. It implies culture of the bacteria on different selective and non-selective media, and characterization of the strains. This identification was managed through 16s rRNA PCR, known as the most common housekeeping genetic marker in bacteria. At the end, we would test the database directly on fabrics stained with wine, or only anthocyanin, looking for colour degradation.

The main advantage is here to select potential useful bacteria from soil, isolate them, and screen all of them, on fabrics, thanks to a high throughput assay. Nevertheless, it implies a selective bias, as some interesting microorganisms may not grow in the media we chose, or cannot compete with other microorganisms present in the soil.
To tackle this problem, we also followed another approach:

• We screened our samples for a microorganism growth directly on anthocyanin-enriched media, and for anthocyanin degradation by absorbance measurement. The idea is here to screen for bacteria that could use anthocyanin as their only carbon source, and thus degrade it, or that could simply metabolize it. After identification of potential interesting microorganisms, we would isolate them, characterize them, and then test them on stained fabrics.

Therefore, the main point of our team is to put in evidence already existing anthocyanin-degradation metabolism in nature, so that we could isolate the enzymes, and potentially optimize them thanks to the binding domain team results.

Week 27th June - 3rd July

The first step is to define a systematic protocol for the selection of microorganisms from our samples.
It includes :

• Determine samples conditions of collection, storage, and treatment before microbes culture.
• Determine the selective and nonselective media we will use for culture and the number of dilutions per samples.
• After restrike and isolation, determine an efficient way to characterize the strains.
• For the second approach, where we test directly an anthocyanin-degradation natural metabolism, define the controls.

Therefore, during this week, we focused our work reading articles dealing with bioremediation and isolation of bacteria from soil samples.
Nonetheless, we rapidly had to tackle the following issue: Such approaches implies protocols of microorganisms culture on anthocyanin enriched media or even as a single carbon source. Or, anthocyanin isolation and purification is hard to achieve, and consequently, this chemical compound is really expensive. We couldn’t afford to buy it in high quantity.
Considering this problem, we decided to take a deeper look in the anthocyanin chemical structure, so that we could potentially find a cheaper substitute molecule for our assays.
Anthocyanin is a phenolic compound which belongs to the Flavonoid family. Flavonoids have a basic structure of C6–C3–C6. Depending on their structures, flavonoids may be classified into about a dozen groups, such as chalcones, flavones, flavonols and anthocyanins.


If we take a deeper look in the anthocyanin structure, we observe that it’s composed of :

• a chroman ring
• an additional aromatic ring on C2

There are 31 different monomeric anthocyanins known. They differ from number and position of the hydroxyl and/or methyl, ether groups.
But, 90% of the naturally occurring anthocyanins are based on only six common anthocyanidins. among them :

• Cyanidin,the major form in nature
• Malvidin, the most commonly found in wine

As we can see in this figure, they only differ by their cycle B groups, so the chroman ring remains unchanged.
Therefore, we believe that enzymes able of degrade a lot of different types of anthocyanins would preferably attack the chroman ring structure as it is well conserved among the family.
Thus, we thought that the ideal cheap substitute molecule should present a very similar basic structure.

After some researches, quercetin, a flavonol molecule, appeared to be a good competitor.
We can in fact see that the only difference remains in the presence of a carbonyl group in 4. Another advantage is that Quercetin is present in Wine, and is partly responsible for its color. Thus, even though the assays may find some enzymes able to degrade specifically quercetin instead of anthocyanin, we should have a decrease in the wine stain color intensity. In addition, because of the co-pigmentation chemical interaction between quercetin and anthocyanin, degrading quercetin could also have an impact on anthocyanin stability.

For these reasons, quercetin was chosen as our substitute molecules for running the microbiological assays.

However, in parallel, we decided to develop a protocol for purifying anthocyanin from grapes, so that we could also tests our microbes directly on anthocyanin. (See the Anthocyanin section for the evolution of the protocol).
Waiting for vineyard samples, we went to Cochin Port Royal’s garden next to our lab, and took some 15 mL of soil.

Week 11th - 17th July

Church paper

This paper is the source of inspiration as he describe the isolation of antibiotic degrading bacteria from soil samples. We decided to try to reproduce it. To isolate the bacteria the researcher used medium with antibiotics as the only carbon source. The main concern was carbon contamination from the soil.

To avoid this contamination the they inoculated the samples in a SCS (single carbon source) liquid medium. They let it grow seven days at 22°C then use the broth to inoculate a new SCS liquid medium. This step is repeted two more time. Then the culture broth is plated on SCS plate and the degrading bacteria isolated. This permit the consumption of all the carbon during the successive liquid cultivation steps and the death of the bacteria that do not degrade the antibiotic.

This protocol have some issues.

• It take 21 days before plating to isolate the microorganisms, this is very long.
• If an organism can degrade anthocyanins but cannot use it as a carbon source this organism is not isolated.

The regular technique to isolate microorganisms from the soil is to dilute and directly inocuate the samplese on plate. So we decided to test if all these steps are necessary. We decided to reproduce this experiment but to plate 4 times instead of one. Plating the inital samples and after each period of growth in liquid medium.

Filtration of the soil sample

A member from the protein group told us that he used a different protocol to isolate a toluene degrading bacteria. To remove the carbon contamination from the soil he filtered the sample with a 0.22µL filter. Then he cultivated the filter in a liquid medium before plating. We decided to test this protocol.

Screening assay design

It seemed less and less probable that we would obtain anthocyanins without carbon contamination and we needed to prepare SCS medium. S we decided to use the colorant quercitin instead of anthocyanin and we ordered it.

Week 18th -24th July

Week 25th -31th July