Week 27th June - 3rd July

This week we were focused on having a protocole for bacteria isolation.
Our aim is to select some strains that are able to degrade wine pigment. The next step is to characterise these strains (thanks to 16s DNA) to have a phylogenetic tree of species degrading wine pigments. At the end, we want to find the gene and the enzymee that are responsible for wine pigment degradation.
In order to select some strain degrading wine pigment, we are using a very simple thought: Stains are organic. Microbes digest organic matter. Therefore, microbes can remove stains. We immagine having a media with the stain and some microbes. If the microbes grow, we isolate them and we know that they were able to degrade our stain.
We designed an experiment thank to the article "Bacteria subsisting on antibiotics" where the autors are using a M9 liquid media with a pure carbon source (in the paper antibiotics). In that case, the grow could only be explained by the utilisation of the single carbon source. But for our experiment, we need a pure carbon source.
But Anthocyane pigment are way too much expensive to buy. We will need for our media about 1g/L and 1mg of malvidin (Anthocyane pigment the most present in wine) is about 130€. But, (For more detail check Anthocyane section)

Week 4th - 10th July

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