Difference between revisions of "Team:Paris Bettencourt/Notebook/Microbiology"

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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. Science2012

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 are creating a bacterial 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 color degradation. We also plan to build a phylogenetic tree based on our database that would help us understanding how related are the species able to degrade anthocyanin.

The main advantage here is 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 here is to screen for bacteria that could use anthocyanin as their sole 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 streak 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 cannot 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.
Flavonoid chemical structure

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).

Week 11th - 17th July

We are looking into the microbes that could be able to degrade quercetin.
Because we want a high diversified database, we asked iGEM team from all around the world to send us soil,grape and grape's leaves samples.

At the end of the competition, we got samples from Melbourne and Macquarie iGEM teams in Australia, from Valencia iGEM team, from Barcelona iGEM team from INSA-Lyon iGEM team, but also from Namibia, Croatia, Algeria, from Cochin and Clos Montmartre in Paris and from Grignon and Suresnes in France.
Samples from Lyon iGEM team

Also, we are looking into some human gut strains for their quercetin degradation ability.
Indeed, wine is know for its cardiovascular protect effect. And we figured out that the compound enabling this effect is actually phenol compound.
On top of that, quercetin and anthocyanin seems to be the most powerful wine antioxydant, and mostly explain this cardiovascular protector effect .
The epithelium from the gut is not able to digest the flavonoid and to cleave it into phenol compound. That's why it is higly probable that the microbiome is responsible for its degradation.
If we were able to isolate some bacteria from human feces on quercetin plate, we may have some very interesting results.
The problem is that we need an authorization to work with human sample. We sent a request for it but it may take a lot of time, with the hope of a quick answer.
Zhang Z, Peng X, Li S, Zhang N, wang Y, Wei H (2014) Isolation and Identification of quercetin Degrading Bacteria from Human Fecal Microbes. PLoS ONE 9(3): e90531. doi:10.1371/journal.pone.0090531

In the same way, It would also be very interesting to work with herbivorous animals, as they eat a lot of flavonoids, responsible for many plants color.
So we can expect that some microbes isolated from these animals' gut could also degrade theses flavonoids into phenolic compounds.
But once again we need some authorizations to work on animals samples.

Week 17th - 25th July

We discovered Church's article, describing the isolation of antibiotic degrading bacteria from soil samples. It appears that the goal was very similar to ours, the only difference remaining in the nature of the molecule.
As they wanted to isolate bacteria that can grow on antibiotics as a unique carbon source, their main concern was carbon contamination from the soil sample.

To avoid this contamination 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 repeated two more times. Then the culture broth is plated on SCS plate and the degrading bacteria is isolated. In these conditions the carbon from the soil is consumed during the first cultivation steps and the bacteria that cannot use antibiotics as a carbon source do not survive during the next cultivation step.

However, this protocol seems to have some issues.

It take 21 days before plating to isolate the microorganisms, this is very long.
Microorganisms that can degrade quercitin without using it as a single carbon source won't be selected.

As we found in several papers, the regular technique to isolate microorganisms from the soil is to dilute the sample on PBS and directly plate on agar. A dilution of 10^(-1) correspond to a dilution of 1g of soil in 9 mL of PBS.

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. In fact, yo 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.

We filtrate a solution of soil sample under vacuum.
Then we place the filter (with the microbes stuck in it without carbons sources) in an erlenmeyer with some M9 + Quercetin. And we waited for a few days.
We had the same results as the protocole with soil sample diluted at 10^-3.
And as you can see in the picture, the green color is disappearing and some fungi is growing in the media.
Filtration of soil sample to avoid carbon contamination

Filtration of soil sample to avoid carbon contamination

Screening assay design

In church lab's article, they cultivate the bacteria at 22°C and pH 5.5 . We found that pH 5.5 inhibit most bacterial growth so we decided to stay at a neutral pH.
We did not have an incubator that could be set at a temperature of 22°C, after a discussion with our advisors and a microbiologist from Cochin hospital it was decided that 30°C should be sufficient for our experiement. Some bacterial species would not survive at 30°C but a sufficient number would, and they may grow faster at 30°C than at 22°C.

We decided to chose 5 dilutions, from 10^(-2) to 10^(-6). Instead of only plating at the end of the 3 liquid cultivation, we decided to plate 100 μL of medium just after inoculation and at the end of each cultivation. The goal of this experiment is to know the dilution needed and to test the impact of the several liquid medium cultivation steps.

We decided to chose 4 different medium (for broth and plate).
-M9 : a medium with salts but no carbon source, this is our negative control medium, if something grows on that medium, it means that there is a carbon contamination.
-M9 glucose (M9 G) : M9 salts plus 1g/L glucose, this medium is our positive control, if nothing grows on it, it means that there were no bacteria in the medium.
-M9 quercetin (M9 Q): M9 salts plus 1g/L quercetin (similar condition than in Church's article), this is the medium where we want to isolate bacterias.
-M9 glucose quercetin (M9 GQ) : this medium is necessary to test if quercetin is toxic for microorganisms. Indeed if we have less growth than in M9 glucose it could mean that quercetin is toxic.

As it is done in church's protocol the incubation time between each cultivation step is 7 days.

Week 25th - 31th July

Quercetin medium preparation

As soon as the quercetin was purchased from Sigma®, we decided to start the assay. However it was found that unlike anthocyanin, quercetin was mostly insoluble in water. This is a major issue for medium preparation as the organisms could not be able to degrade an insoluble molecule in a liquid broth.
But it as also a problem for quercitin quantification, as we thought at the beggining that we could measure the degradation of quercetin with the absorbance, which is not possible if quercitin isn't soluble.

We tried to solubilize quercetin at different concentration in water and ethanol : 1g/L, 0.1g/L, 0.01g/L and 0.001g/L . We did not succeed in dissolving totally the quercetin, so an absorbance assay was impossible.
However, even at 1g/L the liquid was a little yellow, so quercetin was at least slightly soluble.
We thought that the little quantity of soluble quercetin could be enough for growth, as long as the medium is agitated and the quercetin constantly solubilized during the consumption.
So we decided to start the assay anyway. Me managed to have relatively homogenous plate.

Results after two days

The control plates with M9 showed growth until 10^(-3), that means that if we want to avoid carbon contamination in plate, we need to dilute the samples at 10^(-3) at least
The plate with M9 G always showed growth. Even in the more diluted samples there was microorganisms.
The plates with M9 QG showed less growth than the plate with M9G and there was no growth after a dilution of 10^(-3), it could mean that quercetin is toxic for some organisms.

This part of the experiment seemed compromised as there was no growth in the condition M9Q at a dilution were there was no carbon contamination (below 10^(-3))

Growth results in tubes:

M9 growth with a dilution of 10^-2, the carbon contamiation is avoided with a dilution of 10^-3 and below.
M9 G groth in every tube, there is always microorganisms in the samples.
M9 Q and M9 QG, it is difficult to say, the quercitin is precipitated and it seams that the experiment is a failure, that the quercetin cannot be used in liquide medium, maybe because the agitation is too low.

Week 01th - 7th August

Results after a week in solid medium

The results were surprising. In the plates with dilutions of 10^(-2) and 10^(-3), with or without glucose, we could see transparent circles. The quercetin seemed to have disappeared. In the middle of these circles, there were always some microorganisms that looked like filamentous fungi, with the hyphae in the middle and spores in the whole circle.
Show screening

It is to be noted that it is written that the dilution is 10^(-1). This is an annotation mistake. At the beginning we did not counted the initial dilution of the soil sample in water. We have considered that our original sample was 1g of soil in 9mL of water.

Results after a week in liquid medium

With dilution of 10(-2) and 10(-3), in quercetin glucose and in M9 quercetin, there was fungal growth and as the medium was less yellow, it seems that quercetin was partly degraded.
Blank in tubes

Tube with quercitin
Better show screening

Tube with fungal growth (dilution 10^(-2),medium M9Q)
As our laboratory didn't have at this moment a working experience with fungi, we decided to keep the plates at 4°C before working with them, hoping to find bacterias meanwhile.

Week 8th-14th August

Filamentous fungi lab practice

We looked for protocol to work safely with fungi. We found a quite complete fungi protocol book "Laboratory protocol in fungal biology" (M. Ayyachamy and al). There was a safety part. The more important was to prevent spore contamination, knowing that ethanol is not sufficient to kill fungal spores.
A way to disinfect the hood needed to be found. It was said in the book to disinfect with bleach between every experiment and , at the end of the day, to disinfect during 20 minutes with a strong disinfectant like those used in hospitals.

We were able to obtain the disinfectant SURFA’SAFE preminim of the laboratory Anios from the microbiologist of Cochin. It was decided with our advisor to disinfect with bleach between each work with fungi.
Disinfect during 20 minutes with anios was performed each time a member had finished to use fungi.

Quercetin analysis

We still tried to solubilize quercetin. In fact, quercitin was almost insoluble, even at a concentration of 1mg/L, the quercetin precipitated. We tried to heat it but after 1 hour, the quercetin was still not solubilized.
We thought that the solubility of the molecule was of 200g/L so we were very surprised. We looked again on Sigma Aldrich information and that was actually the solubility in DMSO, an organic solvent, which explain our negative results.

The insolubility of quercetin didn't seem to be a real problem for the microorganisms, both in liquid and solid medium. However, it was still a problem for the degradation quantification.
Another problem was the separation between the cells and the quercetin. We tried to filter a sample of water with quercetin with a 0.22 um² filter. All the pigment stayed fixed to filter. The pores were much bigger than the quercetin. It seemed that the quercetin has a strong affinity to the membrane.

As the quercetin is mostly insoluble in water, a centrifugation was a way to remove the cells and the pigment from the broth. All we needed, then, was a medium were the quercetin was soluble, preferably not an expensive and toxic organic solvent.

Week 15th-21th August

Fungal strains collection

We had all we needed to work with filamentous fungi. We isolated the identified strains and inoculated them in Sabouraud Dextrose Agar, a common plate for filamentous fungi culture.
After 4 days the plates were covered of mycelium. Then, we inoculated them on M9Q agar Plate and in M9 plates.

Test in liquid medium

The isolated fungi were tested in 3 liquid medium :

M9
M9 + 1g/L quercetin
M9 + 1g/L quercetin + 1g/L glucose

There was also a blank without fungi

Quercitin analysis

We wanted to see if the disappearance of the color in plates could be due to a pH modification. So we took 1g/L of quercetin and changed the pH with hydrochloric acid. There was no color change at a pH of 1.5.

Then we added base and there was a change. Around a pH of 12 we could see a solubilization of quercetin. After a pH of 12.5, quercetin was completely solubilized. The color was orange instead of yellow. There was no color change between a pH of 12.5 and 13.5.

We had solubilized quercetin. We managed to solubilize up to 50g/L of quercetin in alkaline water.

We measured the absorbance spectrum and we could see that the maximum of absorbance was at 315nm.

Quercetin absorbance at pH 12

To found the link between quercetin concentration and absorbance, we did a calibration.
calibration quercetin

The R² is good, that confirm that the calibration is working. We have a relation between absorbance and concentration : A = 56,767*C, that mean that C=1/(56,767)*A

Finally C = 0,176*A

Week 22th-28th August

Result of the previous assay

There was growth on M9 glucose and M9 quercetin glucose for plates with dilutions of 20^(-2) and 10^(-3). However, the variability was much less important. No quercetin degradation organisms were found. This might be due to the osmotic choc caused by the dilution of the samples in distilled water. We will remember not to repeat this mistake.

Third assay

We did a third assay with a soil sample from Croatia. The conditions were the same than for the two first assays (with PBS and not distilled water !).

Fungal isolation

There was still growth on M9Q plates, we could still see the circles of quercetin degradation. It was confirmed that the strains have been isolated.

Week 29th August-4th September

Fourth assay

We chose to make a simple selection process:

For that, we put some soil sample at 10^-3 of dilution in a single carbon source liquid media. The only carbon source is Quercetin at 1g/L.
We waited for 4 days with the falcon tubes in the shaking incubator at 30°C. In an other hand, we made an other experiment with two carbon sources: Quercetin and Glucose both at concentration of 1g/L.
The idea was to give a chance to some strains that are not maybe able to use Quercetin as a carbon source, but able to destroy this coumpound with some other pathways
By providing them glucose, we could maybe maintain them alive and in the meantime theses strains would be able to degrade Quercetin.

We also had a negative and a positive control.
The negative control was M9 Quercetin without any microbes.
The positive control was Pseudomonas putida.
We made also some conditions with M9 + Glucose with the positive control, the negative control, and the two different soil samples we assayed.
This was done to have an idea of the consumption of glucose by the different strains, measuring the growth of these bacteria at OD 600 nm.
We wanted to see if the degradation of Quercetin is enhanced or inhibited by the presence of glucose.

Quercetin degradation

Surprisingly, Glucose didn’t have the effect we expected.
Pseudomonas was slower but almost unaffected in its degradation of Quercetin while, the microbes from the two soil sample were not able to degrade Quercetin!
We thought that the diauxie phenomena described by Monod, could explain these results.

It is important for us because it’s a way of understanding how the strains are using the Quercetin: is it used as a carbon source via the carbon cycle for growth of the strains or, is it used by another pathway that do not permit the growth of the microbe but is effective in the degradation of the Quercetin (for a toxic compound for instance)
The only solution we found to solubilize quercetin was putting it at pH 12, which would obviously have an impact on bacterial growth.
Thus, this prevent us of doing a kinetic experiment to see the evolution of the growth of a strain with quercetin and glucose at the same time by measuring OD600nm.
Nevertheless, we tried to make an experiment to see if there was a glucose repression with Pseudomonas when we put at the same concentration (1g/L) Glucose and Lactose.

Pseudomonas Kinetic Glucose+Lactose

As you can see on the graph M9+P+G+L (M9+Pseudomonas+Glucose+Lactose) there is no shift in the growth curve of Pseudomonas in presence of Glucose and Lactose.
That means there is no Glucose repression. Further reading of paper confirmed us that there is no Glucose repression for Pseudomonas putida.

We tried to find a new method to measure the growth of strains in liquid media in the presence of Quercetin by measuring absorbance at 600nm.
We tried to solubilize Quercetin in mineral oil and make an emulsion with a two phase solution: Quercetin in the oily phase, Strains in the water phase.

As you can see, when we begin to shake the emulsion, the separation in two phase is not perfect and Quercetin pass through the aqueous phase.

We made some plating at different time: time0, time2, time4.
The idea was to explain the absence of bacteria in our experiments. Indeed, when we were making theses experiments, we always had at the end fungi at the end.
But we should have also some bacteria because bibliography shows that some bacteria are able to degrade Quercetin. By plating at different time we hope to see the difference of diversity of microbes in the plate.
Unfortunately, we were not able to observe some bacteria on Quercetin agar plate. In fact, Quercetin is green and it’s hide the bacteria that are growing on the plate.
During two days we didn’t see any growth of microbes and at day 3, fungi appears.
We have to keep in mind also that fungi, thanks to micellium are able to search for nutrients in the plate while bacteria are not. There is a sort of competition between bacteria and fungi for the use of Quercetin.

Above: two fungi isolated on M9 Quercetin agar plate. We can easily see a clear halo that mean the Quercetin was removed from the plate by the fungi.

Result of the third assey

A fungi was isolated from the plate M9Q 10^(-3) dilution and another from M9Q glucose 10^(-2) plate. Strains labelling and conservation The strains were given a database name and saved at -80°C in 30% glycerol
• FS_F1 : from cochin M9 quercetin plate 10^(-1) dilution
• FS_F2 : from cochin M9 quercetin plate 10^(-2) dilution
• FS_F3 : from cochin M9 glucose quercetin plate 10^(-1) dilution
• FS_F4 : from cochin M9 glucose quercetin plate 10^(-2) dilution
• FS_F5 : from Croatia M9 glucose quercetin plate 10^(-2) dilution
• FS_F6 : from cochin M9 tube plate 10^(-1) dilution
• FS_F7 : from cochin M9 tube 10^(-2) dilution
• FS_F8 : from cochin M9 tube 10^(-1) dilution
• FS_F9 : from cochin M9 tube 10^(-2) dilution
• FS_F10 : from Croatia M9 glucose quercetin plate 10^(-2) dillution

Week 5th-11th September

Quercetin degradation assey

Separation in two project

At this point we really started to work on the first approach, and we combined it to the second one.

The goal of the first approachis making a huge library of strains that were isolated from soil sample.
We would like, from a given soil sample, to isolate as much of bacterial diversity as we can.
Then we could test each strain on Quercetin liquid media and report Quercetin degradation.
Thanks to our collaboration with other iGEM team, we got samples from mant places around the world.

At the end we would have a library with some strains that are able to degrade Quercetin and some others that are not.
We could quantify the strength of the degradation and also make a link between the species that are able to degrade the Quercetin to make hypothesis about a common enzyme responsible for this degradation, through the creation of a phylogenetic tree.

To achieve that, here are the step of the protocol:

We plate the soil samples at 10^-2 and 10-3 dilutions (we choose these dilutions thanks to previous experiments) on non-selective agar media (LB, TSA, M9Glucose, M9) but also on selective media (FTO for micrococcus, Agar from Mossel for Bacillus Cereus, M9 Quercetin).
We then incubate for one or two days at 30°C.
When we have some colonies, we streak some that seems differents on LB or TSA plate in order to easy their growth. Once the streak bacteria grew enough, we give them a name to store them in the library.

Then we make an overnight culture in Tryptic Soy Broth and systematically, we characterise our strain with a 16s rRNA PCR from colonies streaked on non-selective media plates.

When the overnight culture is over, we inoculate 100ul of it directly in a 5ml M9 1g/L Quercetin falcon tube in triplicate.
We also make two glycerol stocks: One at -80°C and the other at -20°C (to be reused more easily).

For the assay on quercitin liquid media, we are quantifying Quercetin degradation.

To see if there was a degradation, we need to use a negative and a positive control.
The negative control is just M9 + Quercetin. The positive control is M9 + Quercetin + Pseudomonas Putida.
Indeed, some article show that Pseudomonas is a good candidate for Quercetin degradation. The iGEM Evry team gave us this strain to use it for our assay.
We made an experiment to check how long does pseudomonas needs to degrade Quercetin.
We found that 4 days are enough to see a significant decrease in Quercetin absorbance. But to give a chance to every strains we will assay, we decided to make a 6 days experiment.

Quercetin degradation with Pseudomonas Putida KT2440

Quercetin degradation with Pseudomonas Putida KT2440

Week 12th-18th September

We designed the experiment with a Quercetin quantification at Time 0 and at Time 6 to see the evolution of Quercetin concentration.

We used Pseudomonas Putida K2440 given from Evry as a positive control.
We made two quantifications:

At T0 to make sure that each tube had the same amount of Quercetin.
And at T6 because it’s enough to see a significant decrease in Quercetin concentration if there is a strain able to use it as a carbon source.

All culture were made in triplicate.
To present data, we made a ratio of the absorbance at T6 over the absorbance at T0 for every culture. Then we average the three values. And we are able to calculate the standard deviation.
The values in y axis are a percentage of remaining Quercetin.
Quercetin degradation experiment

Here you can see the T6 sample. We took 100µl from the 5ml culture after vortexing it and we put it in a eppendorf tube. Then we add 900µl of NaOH 0.5mM solution at pH=12.
You can see the difference on each tube depending of the % of Quercetin remaining.

Quercetin degradation strain 35 to 50

As you can see, none of the strains are able to degrade Quercetin except our positive control.

The issue is that these strains were collected from M9 Quercetin plates, so at least we were expecting some Quercetin degradation.
We suggest that it was a problem with the dilution factor. Indeed 10^-3 is maybe not enough. The other problem is that it is quite hard to streak small and isolated colonies from these plates, because of the quercetin green color on plate. Thus, we are just able to see big colonies.
The other important point is that some strains are not able to use Quercetin as a carbon source to grow. These strains won’t be selected with this technique using a single source carbon. But these strains maybe able to degrade Quercetin as a substrate for other metabolism.
Here are the first 16S PCR we did for strains 52 to 104. We have to notice that 16S rRNA is present in every bacteria, which means that this PCR is really sensitive : if there is contamination of the strain, usually e.coli in a lab, it's 16s rRNA can be amplified instead of the desired strain's.
Knowing that, we did numerous negative control to ensure that our PCR were pure, and that no contamination interfere with our sequencing procedure, following exactly the same PCR protocol but without adding bacteria to the mix. Thus, all the PCR were maid under the hood, which was anyway essential for our safety, as we were working with unknown bacteria isolated from soil, in order to identify them
All the PCR were made at the same time which explain why the negative control don't necessarily appear in the same gel of all their related PCR products. Of course, it is essential to check. 16S PCR strain 52 to 65

C stands for control : here, you can see that they are completely negative, which means no contamination should have interfered with our samples.

Week 19th-25th September

For the next assay, we chose to increase the amount of cells in the media.
We inoculate 500 µl instead of 100 µl of overnight culture in 5 ml of M9 Quercetin liquid media.
Also, concerning plating, we noticed that we most of the time had more or less 4 species of bacteria on non-selective agar. That is annoying because our aim is to have a maximum of bacterial diversity for the database.

Quercetin degradation strain 52 to 74

Yellow plots are some contamination with some fungi in it. But we have at least 3 strains that seems to be very efficient in the degradation.
We took these strains and we plate them from the liquid media to some agar plate and then we re sequenced them to make sure they are the same as those previously tested.

Quercetin degradation strain 75 to 95

So as shows this histogram, we have some good results with E.coli K12 (a strain known to degrade Quercetin but in a different pathway as Pseudomonas Putida.
The strains B1 and B2 are strains that were taken from Anthocyanin degradation assay.
The strains number 75 seems to be very promising.

Quercetin degradation strain 96 to 117

The inoculation was the same as last assay (500ul of overnight culture).
The important thing to notice is that the strains 96, 97, 98, 99, 102, 103, 104 were taken from M9 Quercetin Plate.
We were expecting a degradation...

One reason of this failure maybe that the overnight culture were not enough fresh. Indeed, we had some troubles to organize ourself to test so many strains.
Some overnight culture were 5 days old when we assayed them.
In fact, our 6 incubators are all busy because of the assay, so we have to wait until one is free to begin the assay.
We will organize ourself better next time to avoid this kind of problem.

Quercetin degradation strains 118 to 136

Quercetin degradation strains 118 to 136

For this assay we had one contamination and also no degradation from the other strains.

16S PCR strain 119 to 132

In this experiment we tested also the strains from F1 to F8 that were isolated on Quercetin plate. As you can see, only F4 give a good result with the liquid experiment.
Strains 118 to 136 come from non selective plate (LB agar, Tryptic Soy Agar, M9 agar) Quercetin degradation strain 137 to 153

For this experiment, we tested also 5 E. coli strains that were designed by the Protein group to express some enzyme responsible for Anthocyane degradation. We had no results with these strains but there is maybe a problem of enzyme secretion. We should restart the experiment with cell extract.

Week 26th September-2th October

For this experiments, the strains come from Australia. We isolated them on non selective agar plate.

Fungal identification

The standard sequence used to identify fungi is the ITS sequence. Amplify it we need to purify fungal DNA then do a PCR with ITS 1 and ITS 4 primer. Fungal DNA purification
We tried to purify fungal DNA, the experiment failed because at a moment open tubes fall and the fungal DNA was lost.

Week 9th-16th October

Quercetin degradation strains 179 to 200

Quercetin degradation strains 201 to 222

We isolated a lot of Pseudomonas from strains 201 to 222 that's why we had that much degradation in Quercetin.

Finally with the 9 experiments we made to assay 187 strains, we were able to produce this histogramme.
Quercetin degradation by 183 strains isolated on selective and non selective media

Quercetin degradation by 183 strains isolated on selective and non selective media

To be more clear and avoid confusion with the name of the strains we renamed all the strains we have tested on Quercetin degradation. We named them with a S.number if the strain was isolated on a Selective media (M9 + Quercitin agar). We named them NS.number is the strain was isolated on a Non Selective media.
In this histogram, the strains are rated by chronological order. At the beginning of our experiments, we had only strains isolated on Quercetin that worked on Quercetin degradation.
But then we tested more and more NS strains and we had more positiv results, especially in the end of our experiments when we tested on Australia sample and we had lot of Pseudomonas.

Fungal identification

The fungal DNA was purified. We amplified the ITS region in PCR. The gel electrophoresis showed the sequence in all the strains except the FS_F7 and the negative control.
Assey1

The DNA concentration was quantified :
-negative control : 5 ng/µ
-FS_F1 : 40,4 ng/µL
-FS_F2 : 66,3 ng/µL
-FS_F3 : 62,0 ng/µL
-FS_F4 : 53,0 ng/µL
-FS_F5 : 35,2 ng/µL
-FS_F6 : 23,6 ng/µL
-FS_F8 : 30,0 ng/µL
-FS_F9 : 68,9 ng/µL
-FS_F10: 45,3 ng/µL

As all the concentration were between 20 and 80 ng/µL the DNA was sent to GATC for sequencing

Week 3d October-8th October

Fungal identification

The fungi were identified :
-FS_F1 : Aspergillus fumigatus
-FS_F2 : Aspergillus fumigatus
-FS_F3 : Aspergillus fumigatus
-FS_F4 : Fusarium solani
-FS_F5 : Aspergillus alliaceus
-FS_F6 : Aspergillus fumigatus
-FS_F7 : no identification
-FS_F8 : Aspergillus fumigatus
-FS_F9 : Aspergillus fumigatus
-FS_F10 : Aspergillus niger

So we had 4 different fungi, 6 of our fungi were A.fumigatus, it is to be noted that these 6 fungi all come from the same soil sample.

Quercetin degradation assey

As the standard error bars in the first quercetin degradation assey were really bad we decided to test another time the quercetin degradation after 4 days.
We did a first measure after 2 days :
Assey1

We noted that A.niger was able to degrade quercetin very fast.
We were able to see a difference in quercetin degradation by the different fungi. We were also able to see a difference between the fungi and P.putida.
We wanted to have a picture of each of the fungi in plates so a little ball of mycelium was inoculated on quercetin plates for 3 days
Assey1

We can see the plate becoming transparent where the mycelium have grown.

Week 17th-19th October

We made some kinetic of the most promising strains to see the evolution of Quercetin degradation. All the culture were made in triplicate.
Kinétic of Quercetin degradation

As you can see, there is some variations in the absorbance that makes the curve non linear.
Here we chose not show the standard deviation to make the graph more clear.

To make the graph easier to understand, we chosed to normalized every daily absorbance by the control: Abs(strains)Tx/AbsTx(control).
Thanks to that we get an easy graph to read.
On the Y axis, we can read the % of Quercetin remaining.

We sent to genome sequencing the strains FS_M75(Lysinibacillus), B2(Stenothrophomonas maltophilia), B3(Oerskovia Paurometabola), FS_M121(Microccocus Luteus).
We would like to compare, between theses species, the enzyme commonly responsible for Quercetin degradation.fimage

To finish our project, we assayed two strains FS_M75 and Pseudomonas putida with real wine stain on cotton fabric.

The first experiment was made by putting a circle of cotton into a beaker with wine and then drying it by waiting a few hours.
We had some cells that were in a transparent media composed of M9. This M9 is composed by cells without LB (LB have been removed by centrifugation, cells were re-suspended in PBS).
And we have fabric in M9 without strains and the piece of cotton with the wine stain.

As you can see, the results are not evidential. It seems that our negative control wash the wine stain in an as efficient way as the two other strains!

We redo the experiment but we waited three days until the cotton was enough stained. Then we autoclaved the cotton to make sure there is no others microorganisms. We used the same experimental conditions as before.

We made also an experiment with the platform the assay group designed.
We made three different dilution and we tested once again the ability of Pseudomonas and FS_M75 to degrade wine stain at 30°C.

We used Geneious to generate consensus sequences from each bacteria isolated and sequenced, through de novo alignement between the two chromatogram files (.ab1) provided vy GATC for each sequencing.
Thus, we were able to increase the quality of our sequences, to make them more reliable. We also trimmed them when the quality was too low.
Because of bad quality, or errors in sequencing, some strains of the 187 database were deleted.
Which explain why only 173 bacteria were used to create the phylogenetic tree.
We ran the tree thanks to Geneious software with the following parameters:

The Tree build model chosen was neighbor joining.
Jukes-Cantor was chosen as the genetic distance model
NR_102450 , a cyanobacteria, was chosen as the outgroup
We chose a cost matrix for alignment of 93% similarity (the highest proposed by Geneious)

Pay attention to put every sequences in the same direction (using the Reverse complementary option), as Geneious don't reverse them automatically !
If you forget to do that, tou will have actually 2 similar trees: on assembling the forward sequences, the other one with the reverse ones. Here is the first phylogenetic tree we were able to get. The bold strains with an * are the one that were considered as the most able to degrade quercetin, according to the previous assay. The taxonomy is summed up in the legend.

A.niger kinteics

A.niger degradation kinetics As A.niger was able to degrade all the quercetin in 48h we decided to measure precidly the degradation kinetics. We followed the quercetin degradation for 32h (almost all the quercetin was degraded

Phylogenetic Tree

We could clearly see the exponential aspect of the curb. Almost all the quercetin degradation happened between 16h and 29h. We calculated the degreadation speed between these two moments : V=(C(17)-C(29))/(29-17) = 0,8g/h This value is important to study the impact of a gene on the A.niger’s ability to degrade quercetin. If we had more time we would have done this kind of kinetics on all the fungi to compare their ability to degrade quercetin.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France

+33 1 44 41 25 22/25

igem2016parisbettencourt@gmail.com
2016.igem.org

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+	<div id="topheader">
+	</div>
+	<div id="subheader">
-<!--h1 class="red">Microbiology Group: The Search for Anthocyanin Degradation in Nature </h1-->
+		<table id="ancre">
+			<tr>
+				<td><a href="#ancre">Notebook</a></td>
- <!----------------------- BEGIN SUMMARY BOXES------------------------->
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Protocols">Protocols</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Bibliography">Bibliography</a></td>
-<div class="projtile1">
+			</tr>
-    <h2 class="red" style="text-align:center;">Goals</h2>
+		</table>
-    <ul>
+	</div>
-        <li>To screen soil samples from around the world for microbes that naturally degrade anthocyanin.</li>
+<div id=subheader2 >
-        <li>To identify enzymes linked to the most efficient anthocyanin eaters.</li>
+		<table>
-    </ul>
+			<tr>
+                <td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Assay">Assay</a></td>
+				<td><a href="#ancre2">Microbiology</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Enzyme">Enzyme search</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Indigo">Mission Indigo</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Binding">Binding Domains</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Anthocyanin">Anthocyanin</a></td>
+			</tr>
+		</table>
 </div>
+<div id="ancre2">
+	<div class="input">
-<div style="clear: both;"></div>
+		<h2 class="red">Introduction</h2>
+The microbiology team aims to identify wine stain degrading microorganisms.
+			    <br> <br>
+		        Basing this bioremediation-inspired approach on the two following articles:
+				    <ul>
+					    <li> Bioremediation of phenol by alkaliphilic bacteria isolated from alkaline lake of Lonar, India <br> P.P. Kanekar, S.S. Sarnaik and A.S. Kelkar. <i>Journal of applied microbiology supplement</i> 1999.
+					    </li>
+				        <li> Bacteria Subsisting on Antibiotics <br> Gautam Dantas,  Morten O. A. Sommer,  Rantimi D. Oluwasegun, George M. Church. <i>Science</i>2012
+				        </li>
+				    </ul>
+<br>
+		        We hypothesize that these microorganisms could be preferably discovered in vineyard soil samples.
+		        <br>
+		        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.
+			    <br>
+			    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:
+				    <ul>
+                        <li> We are creating a bacterial 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 color degradation. We also plan to build a phylogenetic tree based on our database that would help us understanding how related are the species able to degrade anthocyanin.
+                        </li>
+                    </ul>
+                The main advantage here is 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.
+                <br>
+<br>
+                To tackle this problem, we also followed another approach:
+                <br>
+                    <ul>
+                        <li> We screened our samples for a microorganism growth directly on anthocyanin-enriched media, and for anthocyanin degradation by absorbance measurement. The idea here is to screen for bacteria that could use anthocyanin as their sole 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.
+                        </li>
+                    </ul>
+                <br>
+<br>
+                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.
+        <h2 class="red">Week 27th June - 3rd July</h2>
+                The first step is to define a systematic protocol for the selection of microorganisms from our samples.
+                <br>
+                It includes :
+                <br>
+                    <ol>
+                        <li>Determine samples conditions of collection, storage, and treatment before microbes culture.
+                        </li>
+                        <li>Determine the selective and nonselective media we will use for culture and the number of dilutions per samples.
+                        </li>
+                        <li>After streak and isolation, determine an efficient way to characterize the strains.
+                        </li>
+                        <li>For the second approach, where we test directly an anthocyanin-degradation natural metabolism, define the controls.
+                        </li>
+                    </ol>
+                <br>
+                Therefore, during this week, we focused our work reading articles dealing with bioremediation and isolation of bacteria from soil samples.
+                <br>
+                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 cannot afford to buy it in high quantity.
+                <br><br>
+                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.
+                <br><br>
+                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.
+                <br>
+                    <center> <img src="https://static.igem.org/mediawiki/2016/c/c9/Paris_Bettencourt-Flavonoid_structure.jpeg" alt="Flavonoid chemical structure" style="width:600px;" align="middle"> </center>
+                <br>
+                <br>
+                If we take a deeper look in the anthocyanin structure, we observe that it’s composed of :
+                    <ul>
+                        <li> a chroman ring </li>
+                        <li> an additional aromatic ring on C2 </li>
+                    </ul>
+                    <center> <img src="https://static.igem.org/mediawiki/2016/9/9e/Paris_Bettencourt-Flavonoid_image.jpeg" alt="Anthocyanin chemical structure" style="width:600px;" align="middle"> </center>
+                <br>
+                There are 31 different monomeric anthocyanins known. They differ from number and position of the hydroxyl and/or methyl, ether groups.
+                <br>
+                But, 90% of the naturally occurring anthocyanins are based on only six common anthocyanidins. among them :
+                    <ul>
+                        <li> Cyanidin,the major form in nature
+                        </li>
+                        <li> Malvidin, the most commonly found in wine
+                        </li>
+                    </ul>
+                <br>
+                As we can see in this figure, they only differ by their cycle B groups, so the chroman ring remains unchanged.
+                <br>
+                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.
+                <br>
+                <b>Thus, we thought that the ideal cheap substitute molecule should present a very similar basic structure.</b>
+                <br>
+                <br>
+                After some researches, Quercetin, a flavonol molecule, appeared to be a good competitor.
+                <br>
+                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.
+                <br>
+                <br>
+                <b>For these reasons, quercetin was chosen as our substitute molecules for running the microbiological assays.</b>
+                <br>
+                     <margin-left> <img src="https://static.igem.org/mediawiki/2016/e/e7/Paris_Bettencourt-Quercitin_image.jpeg" alt="Quercitin chemical structure" style="width:300px;" align="middle"> </margin-left>
+                     <margin-right> <img src="https://static.igem.org/mediawiki/2016/a/a4/Paris_Bettencourt-Anthocyanin_picture.jpeg" alt="Anthocyanin chemical structure" style="width:300px;" align="middle"> </margin-right>
+                <br>
+                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).
+                <br>
+        <h2 class="red">Week 11th - 17th July</h2>
+<br>We are looking into the microbes that could be able to degrade quercetin. <br> Because we want a high diversified database, we asked iGEM team from all around the world to send us soil,grape and grape's leaves samples.<br>
+<br>
+At the end of the competition, we got samples from Melbourne and Macquarie iGEM teams in Australia, from Valencia iGEM team, from Barcelona iGEM team from INSA-Lyon iGEM team, but also from Namibia, Croatia, Algeria, from Cochin and Clos Montmartre in Paris and from Grignon and Suresnes in France.
+<br><center> <img src="https://static.igem.org/mediawiki/2016/3/33/Paris_Bettencourt-File_Samples.jpg" alt="Samples from Lyon iGEM team" style="width:500px;" align="middle"> </center>
+<br>
+<br>Also, we are looking into some human gut strains for their quercetin degradation ability. <br>Indeed, wine is know for its cardiovascular protect effect. And we figured out that the compound enabling this effect is actually phenol compound.<br>
+On top of that, quercetin and anthocyanin seems to be the most powerful wine antioxydant, and mostly explain this cardiovascular protector effect .<br>
+The epithelium from the gut is not able to digest the flavonoid and to cleave it into phenol compound. That's why it is higly probable that the microbiome is responsible for its degradation.<br>If we were able to isolate some bacteria from human feces on quercetin plate, we may have some very interesting results.<br>
+The problem is that we need an authorization to work with human sample. We sent a request for it but it may take a lot of time, with the hope of a quick answer.
+<br>Zhang Z, Peng X, Li S, Zhang N, wang Y, Wei H (2014) Isolation and Identification of quercetin Degrading Bacteria from Human Fecal Microbes. <i>PLoS</i> ONE 9(3): e90531. doi:10.1371/journal.pone.0090531
+<br>
+<br>In the same way, It would also be very interesting to work with herbivorous animals, as they eat a lot of flavonoids, responsible for many plants color.<br>So we can expect that some microbes isolated from these animals' gut could also degrade theses flavonoids into phenolic compounds.<br> But once again we need some authorizations to work on animals samples.
+<br>
+<br>
-<div class="projtile3">
+        <h2 class="red">Week 17th - 25th July</h2>
-      <h2 class="red" style="text-align:center;">Results</h2>
+                We discovered Church's article, describing the isolation of antibiotic degrading bacteria from soil samples. It appears that the goal was very similar to ours, the only difference remaining in the nature of the molecule.<br>  As they wanted to isolate bacteria that can grow on antibiotics as a unique carbon source, their main concern was carbon contamination from the soil sample.
-      <ul>
+                <br>
-            <li>We managed to isolate species from all around the world through iGEM collaborations.
+                <br>
-            <li>186 bacteria were tested for quercetin degradation.
+                To avoid this contamination 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.<br>
-            <li>A 186 bacterial database was built and characterized.
+This step is repeated two more times. Then the culture broth is plated on SCS plate and the degrading bacteria is isolated.  In these conditions the carbon from the soil is consumed during the first cultivation steps and the bacteria that cannot use antibiotics as a carbon source do not survive during the next cultivation step.
-            <li>A phylogenetic tree of 174 different  bacterial species was created .
+                <br>
-            <li>3 bacterias were shown able to degrade anthocyanin
+                <br>
-            <li>Common candidate genes were selected through genome sequencing of 4 different bacterial strains.</li>
+                However, this protocol seems to have some issues.
-      </ul>
+                    <ul>
-</div>
+                        <li>It take 21 days before plating to isolate the microorganisms, this is very long.
+                        </li>
+                        <li>Microorganisms that can degrade quercitin without using it as a single carbon source won't be selected.
+                        </li>
+                    </ul>
+                <br>
+                <br>
+                As we found in several papers, the regular technique to isolate microorganisms from the soil is to dilute the sample on PBS and directly plate on agar. A dilution of 10^(-1) correspond to a dilution of 1g of soil in 9 mL of PBS. <br>
+                <br>
+        <h3>Filtration of the soil sample </h3>
+                A member from the protein group told us that he used a different protocol to isolate a toluene degrading bacteria. In fact, yo 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.
+                <br>
+                <br>
+We filtrate a solution of soil sample under vacuum. <br> Then we place the filter (with the microbes stuck in it without carbons sources) in an erlenmeyer with some M9 + Quercetin. And we waited for a few days. <br>We had the same results as the protocole with soil sample diluted at 10^-3. <br>And as you can see in the picture, the green color is disappearing and some fungi is growing in the media. <br>
+<center> <img src="https://static.igem.org/mediawiki/2016/7/7c/Paris_Bettencourt-File_Filter_strains.jpg" alt="Filtration of soil sample to avoid carbon contamination"  style="width:500px;"align="middle"> </center><br>
+        <h3>Screening assay design </h3>
+                <br>
+                In church lab's article, they cultivate the bacteria at 22°C and pH 5.5 . We found that pH 5.5 inhibit most bacterial growth so we decided to stay at a neutral pH.
+                <br>
+                We did not have an incubator that could be set at a temperature of 22°C, after a discussion with our advisors and a microbiologist from Cochin hospital it was decided that 30°C should be sufficient for our experiement. Some bacterial species would not survive at 30°C but a sufficient number would, and they may grow faster at 30°C than at 22°C. <br>
+                <br>
+                We decided to chose 5 dilutions, from 10^(-2) to 10^(-6). Instead of only plating at the end of the 3 liquid cultivation, we decided to plate 100 μL of medium just after inoculation and at the end of each cultivation. The goal of this experiment is to know the dilution needed and to test the impact of the several liquid medium cultivation steps.<br>
+                <br>
+                We decided to chose 4 different medium (for broth and plate).<br>
+                -M9 : a medium with salts but no carbon source, this is our negative control medium, if something grows on that medium, it means that there is a carbon contamination.<br>
+                -M9 glucose (M9 G) : M9 salts plus 1g/L glucose, this medium is our positive control, if nothing grows on it, it means that there were no bacteria in the medium. <br>
+                -M9 quercetin (M9 Q): M9 salts plus 1g/L quercetin (similar condition than in Church's article), this is the medium where we want to isolate bacterias.<br>
+                -M9 glucose quercetin (M9 GQ) : this medium is necessary to test if quercetin is toxic for microorganisms. Indeed if we have less growth than in M9 glucose it could mean that quercetin is toxic. <br>
+                <br>
+                As it is done in church's protocol the incubation time between each cultivation step is 7 days. <br>
-<div style="clear: both;"></div>
+         <h2 class="red">Week 25th - 31th July</h2><br>
-<div class="projtile4">
+         <h3>Quercetin medium preparation </h3>
-    <h2 class="red" style="text-align:center;">Methods</h2>
-    <ul>
-         <li>Microbiota cultivation
-        <li>Anthocyanin purification
-        <li>Anthocyanin & Quercitin Measurement
-        <li>16S rRNA sequencing
-        <li>Whole genome sequencing
-        <li>Phylogenetic analysis
-    </ul>
-</div>
-<div style="clear: both;"></div>
+                As soon as the quercetin was purchased from Sigma®, we decided to start the assay. However it was found that unlike anthocyanin, quercetin was mostly insoluble in water. This is a major issue for medium preparation as the organisms could not be able to degrade an insoluble molecule in a liquid broth.<br>
+But it as also a problem for quercitin quantification, as we thought at the beggining that we could measure the degradation of quercetin with the absorbance, which is not possible if quercitin isn't soluble.
+                <br>
+<br>
+                We tried to solubilize quercetin at different concentration in water and ethanol : 1g/L, 0.1g/L, 0.01g/L and 0.001g/L . We did not succeed in dissolving totally the quercetin, so an absorbance assay was impossible. <br> However, even at 1g/L the liquid was a little yellow, so quercetin was at least slightly soluble.<br>  We thought that the little quantity of soluble quercetin could be enough for growth, as long as the medium is agitated and the quercetin constantly solubilized during the consumption.<br> So we decided to start the assay anyway. Me managed to have relatively homogenous plate. <br>
+                <br>
-  <!----------------------- END SUMMARY BOXES------------------------->
+        <h3>Results after two days </h3>
+<br>
+<ul>
+Growth results in plate:
+  <li>The control plates with M9 showed growth until 10^(-3), that means that if we want to avoid carbon contamination in plate, we need to dilute the samples at 10^(-3) at least</li>
+               <li> The plate with M9 G always showed growth. Even in the more diluted samples there was microorganisms.</li>
+               <li> The plates with M9 QG showed less growth than the plate with M9G and there was no growth after a dilution of 10^(-3), it could mean that quercetin is toxic for some organisms.</li
+               <li> The plates with M9 QG showed growth until a dilution of 10^(-2) because of the carbon contamination.<li>
+</ul>
+<br>
+ <b>This part of the experiment seemed compromised as there was no growth in the condition M9Q at a dilution were there was no carbon contamination (below 10^(-3))</b>
+               <br>
+               <br>
-<div id=subheader>
+Growth results in tubes:
-<div id="input">
+<ul>
-<h2 class="red">Abstract</h2>
+<li> M9 growth with a dilution of 10^-2, the carbon contamiation is avoided with a dilution of 10^-3 and below.</li>
-<p>
+               <li> M9 G groth in every tube, there is always microorganisms in the samples. </li>
-<b>Anthocyanins, the key pigments found in red wine, are abundant in grapes, berries, flowers and many plants. Like all naturally ocurring metabolites, they eventually biodegrade and re-enter the carbon cycle. In this project, we search nature for enzymatic pathways that can break down anthocyanins into simpler, unpigmented molecules. We reasoned that microbes living in the soil near vineyards were likely to catabolize and consume anthocyanin. Therefore we collected soil samples from 10 vinyards around France, Europe and the world, notably with the help of our fellow iGEM teams. In total we isolated 186 strains through selective and non-selective plating on different media. All of the strains were identified by 16s rRNA sequencing, then characterized for their ability to degrade quercitin, a compound structurally similar to anthocyanin. By phylogenetic analysis, we were able to connect quercitin degradation to specific bacterial phyla and genus including <i>Micrococcus</i>, <i>Pseudomonas</i>, <i>Lysinibacillus</i> and <i>Oerskovia</i>. The most effective strains were further characterized by whole genome sequencing to identify enzymes linked to natural quercitin degradation. By bioprospecting with the help of the worldwide iGEM community, we were able to find the best stain fighting enzymes that nature has to offer.</b>
+               <li>M9 Q and M9 QG, it is difficult to say, the quercitin is precipitated and it seams that the experiment is a failure, that the quercetin cannot be used in liquide medium, maybe because the agitation is too low. </li>
-</p>
+</ul>
-<h2 class="red"> Motivation and Background</h2>
+         <h2 class="red">Week 01th - 7th August</h2>
-<h3>Bioprospecting and Bioremediation</h3>
-<p>
+   <h3>Results after a week in solid medium</h3>
-Bioprospecting is the process of searching nature for genetic information that can be adapted in useful or profitable ways. In recent years, bioprospecting efforts have focused on the search for small molecule pharmaceuticals and other bioactive compounds (Müller, 2016). Bioremediation is the use of living organisms to remove environmental toxins from contaminated areas. Microbes in particular are well known for their ability to degrade organic pollutants like petroleum, pesticides and phenolic compounds. Bioremediation has always been a popular topic in iGEM, producing many notable projects with diverse organisms and applications.</p>
+<br>
+The results were surprising. In the plates with dilutions of 10^(-2) and 10^(-3), with or without glucose, we could see transparent circles. The quercetin seemed to have disappeared. In the middle of these circles, there were always some microorganisms that looked like filamentous fungi, with the hyphae in the middle and spores in the whole circle.
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/e/e2/Paris_Bettancourt-Photo_show_screening.jpeg" alt="Show screening" style="width:500px;" align="middle"> </center><br>
+<center> <img src="https://static.igem.org/mediawiki/2016/a/ac/Paris_Bettencourt-Better_show_screening.jpeg" alt="Better show screening" style="width:500px;" align="middle"> </center><br>
+<br>
+It is to be noted that it is written that the dilution is 10^(-1). This is an annotation mistake. At the beginning we did not counted the initial dilution of the soil sample in water. We have considered that our original sample was 1g of soil in 9mL of water.<br>
+<br>
-<div id="figurebox">
+   <h3>Results after a week in liquid medium</h3>
+<br>
+With dilution of 10(-2) and 10(-3), in quercetin glucose and in M9 quercetin, there was fungal growth and as the medium was less yellow, it seems that quercetin was partly degraded.
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/d/de/Paris_Bettancourt-Blank_in_tubes.jpg" alt="Blank in tubes" style="width:500px;" align="middle"> </center>
+<center>Tube with quercitin<br></center>
+<center> <img src="https://static.igem.org/mediawiki/2016/3/32/Paris_Bettancourt-Fungi_in_tube.jpg" alt="Better show screening" style="width:500px;" align="middle"> </center>
+<center>Tube with fungal growth (dilution 10^(-2),medium M9Q)</center><br>
-<table border="1">
+As our laboratory didn't have at this moment a working experience with fungi, we decided to keep the plates at 4°C before working with them, hoping to find bacterias meanwhile.
-<tr>
+ 	<h2 class="red">Week 8th-14th August</h2>
-<th>Team</th>
-<th>Year</th>
-<th>Project</th>
-</tr>
-<tr>
+   <h3> Filamentous fungi lab practice</h3>
-<td>Leicester</td>
+<br>
-<td>2012</td>
+We looked for protocol to work safely with fungi. We found a quite complete fungi protocol book "Laboratory protocol in fungal biology" (M. Ayyachamy and al). There was a safety part. The more important was to prevent spore contamination, knowing that ethanol is not sufficient to kill fungal spores. <br> A way to disinfect the hood needed to be found. It was said in the book to disinfect with bleach between every experiment and , at the end of the day, to disinfect during 20 minutes with a strong disinfectant like those used in hospitals. <br>
-<td><a href="http://uoleicesterigem2012.blogspot.fr/p/project.html">Polystyrene Biodegradation </a></td>
+<br>
-</tr>
+We were able to obtain the disinfectant SURFA’SAFE preminim of the laboratory Anios from the microbiologist of Cochin.<bt>  It was decided with our advisor to disinfect with bleach between each work with fungi. <br>
+Disinfect during 20 minutes with anios was performed each time a member had finished to use fungi.
-<tr>
+<h3> Quercetin analysis</h3>
-<td>UCL </td>
+<br>
-<td>2012</td>
-<td><a href="https://2012.igem.org/Team:University_College_London">Plastic Republic </a></td>
-</tr>
-<tr>
+We still tried to solubilize quercetin. In fact, quercitin was almost insoluble, even at a concentration of 1mg/L, the quercetin precipitated. We tried to heat it but after 1 hour, the quercetin was still not solubilized. <br> We thought that the solubility of the molecule was of 200g/L so we were very surprised. We looked again on Sigma Aldrich information and that was actually the solubility in DMSO, an organic solvent, which explain our negative results.
-<td>TU-Munich</td>
+<br>
-<td>2013</td>
+<br>
-<td><a href="https://2013.igem.org/Team:TU-Munich">PhyscoFilter</a></td>
+The insolubility of quercetin didn't seem to be a real problem for the microorganisms, both in liquid and solid medium. However, it was still a problem for the degradation  quantification.
-</tr>
+<br>
+Another problem was the separation between the cells and the quercetin. We tried to filter a sample of water with quercetin with a 0.22 um² filter. All the pigment stayed fixed to filter. The pores were much bigger than the quercetin. It seemed that the quercetin has a strong affinity to the membrane. <br>
+<br>
+<br>As the quercetin is mostly insoluble in water, a centrifugation was a way to remove the cells and the pigment from the broth. All we needed, then, was a medium were the quercetin was soluble, preferably not an expensive and toxic organic solvent.
-</tr>
+ 	<h2 class="red">Week 15th-21th August</h2>
-<tr>
-<td>IIT Delhi</td>
-<td>2014</td>
-<td><a href="https://2014.igem.org/Team:IIT_Delhi">Oxide Decontamination </a></td>
-</tr>
-</tr>
+   <h3> Fungal strains collection</h3>
-<tr>
+<br>
-<td>NEFU China</td>
+We had all we needed to work with filamentous fungi. We isolated the identified strains and inoculated them in Sabouraud Dextrose Agar, a common plate for filamentous fungi culture.<br>  After 4 days the plates were covered of mycelium. Then, we inoculated them on M9Q agar Plate and in M9 plates.
-<td>2014</td>
-<td><a href="https://2014.igem.org/Team:NEFU_China/Project">Cadmium Decontamination </a></td>
-</tr>
-</table>
-<p>
-<b>Table 1.</b> Previous iGEM projects using bio-remediation approaches.
-</p>
+   <h3>Test in liquid medium</h3>
-</div>
+<br>
+The isolated fungi were tested in 3 liquid medium : <br>
-<p>
-<br>Anthocyanin is not harmful, but in the context of a stain it is unwanted and so could be considered a target for bioremediation. Therefore a mechanism to degrade anthocyanin could be revealed with a classic bioremediation strategy :
 <ol>
-<li>Select organisms from a contaminated environment, where enzymatic decontamination may have naturally evolved.</li>
+<li>M9</li>
-<li>Isolate pure strains and measure their activity.</li>
+<li>M9 + 1g/L quercetin </li>
-<li>Connect the activity to specific pathways using molecular genetics.</li>
+<li>M9 + 1g/L quercetin + 1g/L glucose </li>
 </ol>
-</p>
+There was also a blank without fungi<br>
-<h3>Anthocyanidin and Quercitin</h3>
+   <h3>Quercitin analysis </h3>
-<p>
+<br>
-In these experiments, we use quercitin as a chemical proxy for anthocyanins. Naturally occurring anthocyanidins are chemically diverse derivatives of a a core flayvlium cation. Plant sources of anthocyanidin carry a range of anthocyanidin pigments substituted at any of up to seven positions, with the relative concentrations contributing to a characteristic color.
+We wanted to see if the disappearance of the color in plates could be due to a pH modification. So we took 1g/L of quercetin and changed the pH with hydrochloric acid. There was no color change at a pH of 1.5. <br>
-Pure anthocyanidins, like malvidin, are expensive (120 EUR for 1 mg) and do not necessarily represent the full chemical diversity of a natural wine stain. Therefore, in this work we use quercitin, a flavonol, as a structural proxy. Quercitn is cheap (40 EUR for 10 g), stable, and can be quantified by absorbance at 315 nm. In follow up experiments, microbes are tested on real wine and on bulk anthocyanins that we extract directly from grape skins.
+<br>
-</p>
+Then we added base and there was a change. Around a pH of 12 we could see a solubilization of quercetin. After a pH of 12.5, quercetin was completely solubilized. The color was orange instead of yellow. There was no color change between a pH of 12.5 and 13.5. <br>
+<br>
+<br>
+<b>We had solubilized quercetin</b>. We managed to solubilize up to 50g/L of quercetin in alkaline water. <br>
+<br>
+<br>
+We measured the absorbance spectrum and we could see that the maximum of absorbance was at 315nm. <br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/a/a1/Paris_Bettencourt-File_Quercetin_pH12_absorbance.jpg" alt="Quercetin absorbance at pH 12" style="width:500px;" align="middle"> </center>
+To found the link between quercetin concentration and absorbance, we did a calibration. <br>
+<center> <img src="https://static.igem.org/mediawiki/2016/1/12/Paris_Bettancourt-Calibration_quercetin.jpg" alt="calibration quercetin" style="width:500px;" align="middle"> </center><br>
+The R² is good, that confirm that the calibration is working. We have a relation between absorbance and concentration : A = 56,767*C, that mean that C=1/(56,767)*A<br>
+<br>
+Finally C = 0,176*A
+ 	<h2 class="red">Week 22th-28th August</h2>
-<div id="figurebox">
+   <h3> Result of the previous assay  </h3>
-<div style="float:left; margin-left: 100px; margin-bottom:10px">
+<br>
-<img src="https://static.igem.org/mediawiki/2016/2/29/Paris_Bettencourt-Malvidin_and_Quercetin_chemical_structure.png" alt="Chemical structure" style="width:360px;">
+There was growth on M9 glucose and M9 quercetin glucose for plates with dilutions of 20^(-2) and 10^(-3). However, the variability was much less important. No quercetin degradation organisms were found. This might be due to the osmotic choc caused by the dilution of the samples in distilled water. We will remember not to repeat this mistake.
-<p>
-</p>
+   <h3> Third assay </h3>
-</div>
+<br>
-<div>
+We did a third assay with a soil sample from Croatia. The conditions were the same than for the two first assays (with PBS and not distilled water !).
-<ul style="margin-left: 450px; margin-right: 100px; margin-bottom:10px">
+   <h3> Fungal isolation</h3>
-<b>Figure 1. Structure and absorbance of malvidin, the most abundant anthocyanidin in wine, and quercetin, a flavonol</b>
 <br>
-<br>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. 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, increasing wine color stability, mainly through π-π stacking between their phenolic cycles. Thus, it leads to the possibility that quercetin degradation could also impact anthocyanin stability.
+There was still growth on M9Q plates, we could still see the circles of quercetin degradation. It was confirmed that the strains have been isolated.
-</ul>
-<br><br>
-</div>
+<h2 class="red">Week 29th August-4th September</h2>
-</div>
 <br>
+<h3> Fourth assay </h3>
+We chose to make a simple selection process:
+<br>
+<br>
+For that, we put some soil sample at 10^-3 of dilution in a single carbon source liquid media. The only carbon source is Quercetin at 1g/L. <br> We waited for 4 days with the falcon tubes in the shaking incubator at 30°C. In an other hand, we made an other experiment with two carbon sources: Quercetin and Glucose both at concentration of 1g/L. <br>The idea was to give a chance to some strains that are not maybe able to use Quercetin as a carbon source, but able to destroy this coumpound with some other pathways <br> By providing them glucose, we could maybe maintain them alive and in the meantime theses strains would be able to degrade Quercetin.<br><br>
+We also had a negative and a positive control. <br> The negative control was M9 Quercetin without any microbes.<br> The positive control was Pseudomonas putida.<br> We made also some conditions with M9 + Glucose with the positive control, the negative control, and the two different soil samples we assayed. <br>This was done to have an idea of the consumption of glucose by the different strains, measuring the growth of these bacteria at OD 600 nm. <br>We wanted to see if the degradation of Quercetin is enhanced or inhibited by the presence of glucose.
+<br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/0/07/Paris_Bettencourt-File_Quercetin_degradation_strain.jpg" alt="Quercetin degradation" style="width:700px;" align="middle"> </center>
+	<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/f/f6/Paris_Bettencourt-File_Quercetin_degradation_strain_M9%2BG.jpg" alt="Quercetin degradation" style="width:700px;" align="middle"> </center>
+	<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/d/d7/Paris_Bettencourt-File_Quercetin_degradationG.jpg" alt="Quercetin degradation" style="width:700px;" align="middle"> </center>
+	<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/a/a3/Paris_Bettencourt-File_Quercetin_degradation.jpg" alt="Quercetin degradation" style="width:700px;" align="middle"> </center>
+	<br>
 <br>
+Surprisingly, Glucose didn’t have the effect we expected.<br> Pseudomonas was slower but almost unaffected in its degradation of Quercetin while, the microbes from the two soil sample were not able to degrade Quercetin!<br> We thought that the diauxie phenomena described by Monod, could explain these results. <br>
+<br>
+It is important for us because it’s a way of understanding how the strains are using the Quercetin: is it used as a carbon source via the carbon cycle for growth of the strains or, is it used by another pathway that do not permit the growth of the microbe but is effective in the degradation of the Quercetin (for a toxic compound for instance)<br>
+The only solution we found to solubilize quercetin was putting it at pH 12, which would obviously have an impact on bacterial growth.<br>
+Thus, this prevent us of doing a kinetic experiment to see the evolution of the growth of a strain with quercetin and glucose at the same time by measuring OD600nm.<br> Nevertheless, we tried to make an experiment to see if there was a glucose repression with Pseudomonas when we put at the same concentration (1g/L) Glucose and Lactose.
+<br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/4/44/Paris_Bettencourt-File_Pseudomonas_Kinetic.jpg" alt="Pseudomonas Kinetic Glucose+Lactose" style="width:700px;" align="middle"> </center>
+	<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/8/82/Paris_Bettencourt-File_Pseudomonas_Control_strains.jpg" style="width:700px;" align="middle"> </center>
+	<br>
+As you can see on the graph M9+P+G+L (M9+Pseudomonas+Glucose+Lactose) there is no shift in the growth curve of Pseudomonas in presence of Glucose and Lactose. <br>That means there is no Glucose repression. Further reading of paper confirmed us that there is no Glucose repression for Pseudomonas putida.
+<br>
+<br>
+We tried to find a new method to measure the growth of strains in liquid media in the presence of Quercetin by measuring absorbance at 600nm. <br>We tried to solubilize Quercetin in mineral oil and make an emulsion with a two phase solution: Quercetin in the oily phase, Strains in the water phase. <br>
+<center> <img src="https://static.igem.org/mediawiki/2016/1/17/Paris_Bettencourt-File_Quercetin_strains.jpg" style="width:700px;" align="middle"> </center>
+	<br> As you can see, when we begin to shake the emulsion, the separation in two phase is not perfect and Quercetin pass through the aqueous phase.
+<br>
+<br>
+We made some plating at different time: time0, time2, time4. <br>The idea was to explain the absence of bacteria in our experiments. Indeed, when we were making theses experiments, we always had at the end fungi at the end.<br> But we should have also some bacteria because bibliography shows that some bacteria are able to degrade Quercetin. By plating at different time we hope to see the difference of diversity of microbes in the plate. <br> Unfortunately, we were not able to observe some bacteria on Quercetin agar plate. In fact, Quercetin is green and it’s hide the bacteria that are growing on the plate.<br> During two days we didn’t see any growth of microbes and at day 3, fungi appears.<br> We have to keep in mind also that fungi, thanks to micellium are able to search for nutrients in the plate while bacteria are not. There is a sort of competition between bacteria and fungi for the use of Quercetin.<br>
+<br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/d/d3/Paris_Bettencourt-File_Fungi_strains.jpg" style="width:700px;" align="middle"> </center>
+	<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/f/fe/Paris_Bettencourt-File_Fungi2.jpg" style="width:700px;" align="middle"> </center>
+	<br>
+Above: two fungi isolated on M9 Quercetin agar plate. We can easily see a clear halo that mean the Quercetin was removed from the plate by the fungi.
+<br>
+<br>
+<h3>Result of the third assey</h3>
+<br>
+<p class="input">
+A fungi was isolated from the plate M9Q 10^(-3) dilution and another from M9Q glucose 10^(-2) plate.
+Strains labelling and conservation
+The strains were given a database name and saved at -80°C in 30% glycerol<br>
+•	FS_F1 : from cochin M9 quercetin plate 10^(-1) dilution<br>
+•	FS_F2 : from cochin M9 quercetin plate 10^(-2) dilution<br>
+•	FS_F3 : from cochin M9 glucose quercetin plate 10^(-1) dilution<br>
+•	FS_F4 : from cochin M9 glucose quercetin plate 10^(-2) dilution<br>
+•	FS_F5 : from Croatia M9 glucose quercetin plate 10^(-2) dilution<br>
+•	FS_F6 : from cochin M9 tube plate 10^(-1) dilution<br>
+•	FS_F7 : from cochin M9 tube 10^(-2) dilution<br>
+•	FS_F8 : from cochin M9 tube 10^(-1) dilution<br>
+•	FS_F9 : from cochin M9 tube 10^(-2) dilution<br>
+•	FS_F10 : from Croatia M9 glucose quercetin plate 10^(-2) dillution<br>
+<br>
+<h2 class="red">Week 5th-11th September</h2>
+<br>
+   <h3>Quercetin degradation assey </h3>
+<br>
-<h2 class="red">Results</h2>
-<h3>Anthocyanin Extraction and analysis</h3>
-<p>Anthocyanins were extracted from <i>Vinis vitifera</i> fruits. It skin was separated from the rest of the fruit and macerated overnight in an ethanol solution with 1% chloridric acid. After maceration, the solution was passed through with a paper filter to eliminate solid material and evaporated at 37°C at 150 rpm. We confirmed the presence of anthocyanin with HPLC and, colour variation with pH.</p>
-<h3>Collection of the soil samples</h3>
+	<h3> Separation in two project </h3>
+	At this point we really started to work on the first approach, and we combined it to the second one.<br><br>
+The goal of the first approachis making a huge library of strains that were isolated from soil sample. <br>
+	We would like, from a given soil sample, to isolate as much of bacterial diversity as we can. <br>
+	Then we could test each strain on Quercetin liquid media and report Quercetin degradation. <br>
+	Thanks to our collaboration with other iGEM team, we got samples from mant places around the world. <br>
+	<br>
+        <br>
+	At the end we would have a library with some strains that are able to degrade Quercetin and some others that are not. <br>
+	We could quantify the strength of the degradation and also make a link between the species that are able to degrade the Quercetin to make hypothesis about a common enzyme responsible for this degradation, through the creation of a phylogenetic tree.
+	<br>
+	<br>
+	To achieve that, here are the step of the protocol:
+	<br>
+	<br>
+<ol>
+<li>We plate the soil samples at 10^-2 and 10-3 dilutions (we choose these dilutions thanks to previous experiments) on non-selective agar media (LB, TSA, M9Glucose, M9) but also on selective media (FTO for micrococcus, Agar from Mossel for Bacillus Cereus, M9 Quercetin).</li>
+<li>We then incubate for one or two days at 30°C.</li>
+<li>When we have some colonies, we streak some that seems differents on LB or TSA plate in order to easy their growth. Once the streak bacteria grew enough, we give them a name to store them in the library.</li>
+For instance, FS_M69 stand for Frank&Stain_Microbesgroup n°=69
+	For a given strain we have the date of the stock, the species once its sequenced, the original agar media from where it was taken, the origin of the strain etc…
+	<br>
+	<br>
+	<center> <img src="https://static.igem.org/mediawiki/2016/2/23/Paris_Bettencourt-File_Strain_DB.jpg" alt="Strain database" style="width:500px;" align="middle"> </center>
+	<br>
+	<br>
+<li>Then we make an overnight culture in Tryptic Soy Broth and systematically, we characterise our strain with a 16s rRNA PCR from colonies streaked on non-selective media plates.</li><br>
+	<li> When the overnight culture is over, we inoculate 100ul of it directly in a 5ml M9 1g/L Quercetin falcon tube in triplicate. </li>
+<li> We also make two glycerol stocks: One at -80°C and the other at -20°C (to be reused more easily).</li> </ol> <br>
+	For the assay on quercitin liquid media, we are quantifying Quercetin degradation. <br>
+	<br>
+	<br>
+	To see if there was a degradation, we need to use a negative and a positive control.<br>
+	The negative control is just M9 + Quercetin. The positive control is M9 + Quercetin + Pseudomonas Putida. <br>
+	Indeed, some article show that Pseudomonas is a good candidate for Quercetin degradation. The iGEM Evry team gave us this strain to use it for our assay.<br>
+	We made an experiment to check how long does pseudomonas needs to degrade Quercetin.<br>
+	We found that 4 days are enough to see a significant decrease in Quercetin absorbance. But to give a chance to every strains we will assay, we decided to make a 6 days experiment.
+	<br>
+	<br>
+	<center> <img src="https://static.igem.org/mediawiki/2016/7/74/Paris_Bettencourt-File_Quercetin_Pseudomonas.jpg" alt="Quercetin degradation with Pseudomonas Putida KT2440" style="width:500px;" align="middle"> </center>
+	<br>
+   	<br>
+<h2 class="red">Week 12th-18th September</h2>
+<br>
+<br>
+	We designed the experiment with a Quercetin quantification at Time 0 and at Time 6 to see the evolution of Quercetin concentration.
+	<br>
+	<br>
+	We used Pseudomonas Putida K2440 given from Evry as a positive control. <br>
+	We made two quantifications:
+<ol>
+<li> At T0 to make sure that each tube had the same amount of Quercetin.</li>
+<li>And at T6 because it’s enough to see a significant decrease in Quercetin concentration if there is a strain able to use it as a carbon source.</li>
+</ol>
+All culture were made in triplicate.<br>
+To present data, we made a ratio of the absorbance at T6 over the absorbance at T0 for every culture. Then we average the three values. And we are able to calculate the standard deviation.<br>
+The values in y axis are a percentage of remaining Quercetin.
+	<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/5/52/Paris_Bettencourt-File_Quercetin_degradation_strains_experiment.jpg" alt="Quercetin degradation experiment" style="width:700px;" align="middle"> </center>
+	<br>
+Here you can see the T6 sample. We took 100µl from the 5ml culture after vortexing it and we put it in a eppendorf tube. Then we add 900µl of NaOH 0.5mM solution at pH=12. <br> You can see the difference on each tube depending of the % of Quercetin remaining.<br>
+	<br>
+	<center> <img src="https://static.igem.org/mediawiki/2016/f/f5/Paris_Bettencourt-File_Quercetin_degradation_strain_35_to_50.jpg" alt="Quercetin degradation strain 35 to 50" style="width:700px;" align="middle"> </center>
+	<br>
+  As you can see, none of the strains are able to degrade Quercetin except our positive control.<br>
+<br>
+The issue is that these strains were collected from M9 Quercetin plates, so at least we were expecting some Quercetin degradation.<br>
+We suggest that it was a problem with the dilution factor. Indeed 10^-3 is maybe not enough. The other problem is that it is quite hard to streak small and isolated colonies from these plates, because of the quercetin green color on plate. Thus, we are just able to see big colonies.<br>
+	The other important point is that some strains are not able to use Quercetin as a carbon source to grow. These strains won’t be selected with this technique using a single source carbon. But these strains maybe able to degrade Quercetin as a substrate for other metabolism.
+	<br>
+Here are the first 16S PCR we did for strains 52 to 104. We have to notice that 16S rRNA is present in every bacteria, which means that this PCR is really sensitive : if there is contamination of the strain, usually e.coli in a lab, it's 16s rRNA can be amplified instead of the desired strain's.<br> Knowing that, we did numerous negative control to ensure that our PCR were pure, and that no contamination interfere with our sequencing procedure, following exactly the same PCR protocol but without adding bacteria to the mix.
+<b>Thus, all the PCR were maid under the hood, which was anyway essential for our safety, as we were working with unknown bacteria isolated from soil, in order to identify them</b><br>
+ All the PCR were made at the same time which explain why the negative control don't necessarily appear in the same gel of all their related PCR products. Of course, it is essential to check.
+        <center> <img src="https://static.igem.org/mediawiki/2016/8/80/16s_PCR_52_66.jpeg" alt="16S PCR strain 52 to 65" style="width:700px;" align="middle"> </center>
+	<br>
+        <center> <img src="https://static.igem.org/mediawiki/2016/2/24/16s_PCR_67-80.jpeg" alt="16S PCR strain 67 to 80" style="width:700px;" align="middle"> </center>
+	<br>
+C stands for control : here, you can see that they are completely negative, which means no contamination should have interfered with our samples.
+<br>
-<p>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 <a href = "https://static.igem.org/mediawiki/2016/b/b7/Paris_Bettencourt_Facture_Proforma.pdf">Facture Proforma</a>, printed out by the sample donor and included in the shipment.
+<br>
+<h2 class="red">Week 19th-25th September</h2>
+	<br>
+	<br>
+For the next assay, we chose to increase the amount of cells in the media.<br>
+	We inoculate 500 µl instead of 100 µl of overnight culture in 5 ml of M9 Quercetin liquid media.<br>
+	Also, concerning plating, we noticed that we most of the time had more or less 4 species of bacteria on non-selective agar. That is annoying because our aim is to have a maximum of bacterial diversity for the database. <br>
+        <br>
+	<center> <img src="https://static.igem.org/mediawiki/2016/e/e9/Paris_Bettencourt-File_Quercetin_degradation_strain_52_to_74.jpg" alt="Quercetin degradation strain 52 to 74" style="width:700px;" align="middle"> </center>
+	<br>
+	Yellow plots are some contamination with some fungi in it. But we have at least 3 strains that seems to be very efficient in the degradation.<br>
+	We took these strains and we plate them from the liquid media to some agar plate and then we re sequenced them to make sure they are the same as those previously tested.
+	<br>
+	<br>
+        <center> <img src="https://static.igem.org/mediawiki/2016/4/47/Paris_Bettencourt-File_Quercetin_degradation_strain_75_to_95.jpg" alt="Quercetin degradation strain 75 to 95" style="width:700px;" align="middle"> </center>
+        <br>
+      <center> <img src="https://static.igem.org/mediawiki/2016/0/06/16s_PCR_81-94.jpeg" alt="16S PCR strain 81 to 94" style="width:700px;" align="middle"> </center>
+        <br>
+<center> <img src="https://static.igem.org/mediawiki/2016/4/41/16s_PCR_95-104.jpeg" alt="16S PCR strain 95 to 104" style="width:700px;" align="middle"> </center>
+        So as shows this histogram, we have some good results with E.coli K12 (a strain known to degrade Quercetin but in a different pathway as Pseudomonas Putida. <br>
+        The strains B1 and B2 are strains that were taken from Anthocyanin degradation assay.<br>
+        The strains number 75 seems to be very promising.
+         <br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/e/ea/Paris_Bettencourt-File_Quercetin_degradation_strain_96_to_117.jpg" alt="Quercetin degradation strain 96 to 117" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/0/0f/Paris_Bettencourt-PCR_105.jpeg" alt="16S PCR strain 105 to 118" style="width:700px;" align="middle"> </center>
+<br>
+The inoculation was the same as last assay (500ul of overnight culture). <br> The important thing to notice is that the strains 96, 97, 98, 99, 102, 103, 104 were taken from M9 Quercetin Plate. <br> We were expecting a degradation...
+<br>
+<br>
+One reason of this failure maybe that the overnight culture were not enough fresh. Indeed, we had some troubles to organize ourself to test so many strains.<br>
+Some overnight culture were 5 days old when we assayed them.<br>
+In fact, our 6 incubators are all busy because of the assay, so we have to wait until one is free to begin the assay.<br> We will organize ourself better next time to avoid this kind of problem.
+<br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/b/b6/Paris_Bettencourt-File_Quercetin_degradation_strains.jpg" alt="Quercetin degradation strains 118 to 136" style="width:700px;" align="middle"> </center>
+<br>
+For this assay we had one contamination and also no degradation from the other strains. <br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/2/22/16s_PCR_119-132.jpeg" alt="16S PCR strain 119 to 132" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/3/30/16s_PCR_120-136.jpeg" alt="16S PCR strain 120 to 136" style="width:700px;" align="middle"> </center>
+<br>
+In this experiment we tested also the strains from F1 to F8 that were isolated on Quercetin plate. As you can see, only F4 give a good result with the liquid experiment.<br>
+Strains 118 to 136 come from non selective plate (LB agar, Tryptic Soy Agar, M9 agar)
+<center> <img src="https://static.igem.org/mediawiki/2016/0/04/Paris_Bettencourt-File_Quercetin_degradation_strain_137_to_153.jpg" alt="Quercetin degradation strain 137 to 153" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/1/1e/Paris_Bettencourt-PCR_137-145.jpeg" alt="16S PCR strain 137 to 145" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/3/39/Paris_Bettencourt-PCR_146-158.jpeg" alt="16S PCR strain 146 to 158" style="width:700px;" align="middle"> </center>
+<br>
+For this experiment, we tested also 5 E. coli strains that were designed by the Protein group to express some enzyme responsible for Anthocyane degradation. We had no results with these strains but there is maybe a problem of enzyme secretion. We should restart the experiment with cell extract.<br>
+<br>
+<br>
-<br>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.
+<br>
-</p>
+<br>
+<h2 class="red">Week 26th September-2th October</h2>
+<br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/5/5e/Paris_Bettencourt-File_Quercetin_degradation_strain_154_to_178.jpg" alt="Quercetin degradation strain 154 to 178" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/7/7c/Paris_Bettencourt-PCR_159-171.jpeg" alt="16S PCR strain 159 to 171" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/b/bf/Paris_Bettencourt-PCR_172-178.jpeg" alt="16S PCR strain 172 to 178" style="width:700px;" align="middle"> </center>
+<br>
+For this experiments, the strains come from Australia. We isolated them on non selective agar plate.
+<br>
+<br>
+<h3>Fungal identification</h3>
+<br>
+The standard sequence used to identify fungi is the ITS sequence. Amplify it we need to purify fungal DNA then do a PCR with ITS 1 and ITS 4 primer.
+Fungal DNA purification <br>
+We tried to purify fungal DNA, the experiment failed because at a moment open tubes fall and the fungal DNA was lost.
+<br>
+<h2 class="red">Week 9th-16th October</h2>
+<br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/b/b1/Paris_Bettencourt-File_Quercetin_degradation_strain_179_to_200.jpg" alt="Quercetin degradation strains 179 to 200" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/e/ed/Paris_Bettencourt-PCR_179-191.jpeg" alt="16S PCR strain 179 to 191" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/4/4b/Paris_Bettencourt-PCR_192-204.jpeg" alt="16S PCR strain 192 to 204" style="width:700px;" align="middle"> </center>
+<br>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/6/6a/Paris_Bettencourt-File_Quercetin_degradation_strain_201_to_222.jpg" alt="Quercetin degradation strains 201 to 222" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/7/7a/Paris_Bettencourt-PCR_205-217.jpeg" alt="16S PCR strain 205 to 217" style="width:700px;" align="middle"> </center>
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/d/d9/Paris_Bettencourt-PCR_218-222.jpeg" alt="16S PCR strain 218 to 222" style="width:700px;" align="middle"> </center>
+<br>
+We isolated a lot of Pseudomonas from strains 201 to 222 that's why we had that much degradation in Quercetin.
+<br>
+<br>Finally with the 9 experiments we made to assay 187 strains, we were able to produce this histogramme. <br>
+<img src="https://static.igem.org/mediawiki/2016/6/66/Paris_Bettencourt-File_Quercetin_degradation_187strains.jpg" alt="Quercetin degradation by 183 strains isolated on selective and non selective media" style="width:120%;" align="middle"> </center>
+<br>
+<br>
+To be more clear and avoid confusion with the name of the strains we renamed all the strains we have tested on Quercetin degradation. We named them with a S.number if the strain was isolated on a Selective media (M9 + Quercitin agar). We named them NS.number is the strain was isolated on a Non Selective media.
+<br>
+In this histogram, the strains are rated by chronological order. At the beginning of our experiments, we had only strains isolated on Quercetin that worked on Quercetin degradation. <br>But then we tested more and more NS strains and we had more positiv results, especially in the end of our experiments when we tested on Australia sample and we had lot of Pseudomonas.
+<br>
+<br>
-<div id="figurebox">
+   <h3>Fungal identification</h3>
-<table border="1">
+<br>
-<tr>
-<th>Country</th>
-<th>Location</th>
-<th>Collector</th>
-</tr>
-<tr>
+The fungal DNA was purified. We amplified the ITS region in PCR. The gel electrophoresis showed the sequence in all the strains except the FS_F7 and the negative control. <br>
-<td>France</td>
-<td>Clos Monmartre Vineyard, Paris</td>
-<td>Our Team</td>
-</tr>
-<tr>
-<td>France</td>
-<td>Cochin Port Royal, Paris</td>
-<td>Our team</td>
-</tr>
-<tr>
+<center> <img src="https://static.igem.org/mediawiki/2016/f/fd/Paris_Bettencourt-gel_electrophoresis.jpg" alt="Assey1" style="width:600px;" align="middle"> </center>
-<td>France</td>
-<td>Vaucluse region’s vineyard</td>
-<td>INSA-Lyon iGEM team</td>
-</tr>
-<tr>
+The DNA concentration was quantified  : <br>
-<td>Spain</td>
-<td>Barcelona</td>
-<td>UPF-CRG Barcelona iGEM team</td>
-</tr>
-<tr>
+-negative control : 5 ng/µ<br>
-<td>Spain</td>
+-FS_F1 : 40,4 ng/µL<br>
-<td>Utiel Requena</td>
+-FS_F2 : 66,3 ng/µL<br>
-<td>UPV Valencia iGEM team</td>
+-FS_F3 : 62,0 ng/µL<br>
-</tr>
+-FS_F4 : 53,0 ng/µL<br>
+-FS_F5 : 35,2 ng/µL<br>
+-FS_F6 : 23,6 ng/µL<br>
+-FS_F8 : 30,0 ng/µL<br>
+-FS_F9 : 68,9 ng/µL<br>
+-FS_F10: 45,3 ng/µL<br>
+<br>
+As all the concentration were between 20 and 80 ng/µL the DNA was sent to GATC for sequencing
+<br>
+<br>
+<h2 class="red">Week 3d October-8th October</h2>
-<tr>
+<h3>Fungal identification</h3>
-<td>Australia</td>
+<br>
-<td>Hunter Valley</td>
-<td>UNSW</td>
-</tr>
-<tr>
+The fungi were identified :<br>
-<td>Australia</td>
+-FS_F1 : Aspergillus fumigatus<br>
-<td>Sydney</td>
+-FS_F2 : Aspergillus fumigatus<br>
-<td>Macquarie 2016 iGEM team</td>
+-FS_F3 : Aspergillus fumigatus<br>
-</tr>
+-FS_F4 : Fusarium solani<br>
+-FS_F5 : Aspergillus alliaceus<br>
+-FS_F6 : Aspergillus fumigatus<br>
+-FS_F7 : no identification<br>
+-FS_F8 : Aspergillus fumigatus<br>
+-FS_F9 : Aspergillus fumigatus<br>
+-FS_F10 : Aspergillus niger<br>
+<br>
+So we had 4 different fungi, 6 of our fungi were A.fumigatus, it is to be noted that these 6 fungi all come from the same soil sample.
+<br>
+<h3>Quercetin degradation assey</h3>
-<tr>
+As the standard error bars in the first quercetin degradation assey were really bad we decided to test another time the quercetin degradation after 4 days. <br>
-<td>Namibia</td>
+We did a first measure after 2 days  : <br>
-<td>Etosha National Park</td>
-<td>Our team</td>
+<center> <img src="https://static.igem.org/mediawiki/2016/7/71/Paris_Bettancourt-Assey_2_48h.png" alt="Assey1" style="width:600px;" align="middle"> </center>
-</tr>
-<tr>
+We noted that A.niger was able to degrade quercetin very fast. <br>
-<td>Algeria</td>
-<td>Alger</td>
+We were able to see a difference in quercetin degradation by the different fungi. We were also able to see a difference between the fungi and P.putida. <br>
-<td>Our team</td>
+We wanted to have a picture of each of the fungi in plates so a little ball of mycelium was inoculated on quercetin plates for 3 days <br>
-</tr>
-<tr>
-<td>Croatia</td>
-<td>Kricke</td>
-<td>Our team</td>
-</tr>
-<tr>
-<td>Israel</td>
-<td>Jerusalem</td>
-<td>Our team</td>
-</tr>
-</table>
-<div style="float:right; margin-bottom:10px;margin-top: -500px; ">
-<img src="https://static.igem.org/mediawiki/2016/f/f8/Paris_Bettencourt-File_Sample_World_microbio.jpg"alt="world_Microbiome" style="width:450px;">
-<p>
-</p>
-<p>
-<b>Table 2 and Figure 2.</b> Location of soil samples<br>collected or obtained by the team.
-</div>
-</div>
-<h3>Preparation of the microbe library</h3>
-<p>
-For safety and to avoid environmental contamination, microbial isolation was performed in a fume hood in a BSL 2 facility. More safety information in provided on our safety page.
-<br>Microbes were isolated from soil samples using either selective or nonselective plating. For the selective plating, M9 agar was supplemented with either quercetin alone or quercetin with glucose to enrich for microbes with the ability to metabolize quercetin.
-<br>The resulting culture was incubated at 30 C for 48 hours, then re-streaked to eliminate potential contamination.
-<br>To maximize library diversity, we preferentially chose colonies with unique morphologies. 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.
-</p>
-<div id="figurebox">
-<div style="text-align:center;">
-<img src="https://static.igem.org/mediawiki/2016/1/1f/Paris_Bettencourt-File_Quercetin_.jpg" alt="Quercetin strains degradation" style="width:900px;">
-<p>
-<b> Figure 3. Quercitin degradation by 189 microbes collected from global soil samples</b> Single colonies were inoculated in M9 minimal medium and grown for 6 days. Quercitin was measured by absorbance. Strains to the extreme left of the figure represent the highest-degrading strains.
-</p>
-</div>
-</div>
-<div id="figurebox">
-    <img src="https://static.igem.org/mediawiki/2016/b/be/Paris_Bettencourt-File_Quercetin_degradation_kinetic.jpg" alt="Quercetin degradation detail" style="width:700px; " >
-<p style="text-align: left;">
-            <b> Figure 4. Quercitin degradation detail</b> The top-performing strains included those isolated from both selective and nonselective media. Strains marked with an asterisk were selected for further investigation.
-        </p>
- </div>
-<p>
-Working with M9 quercetin plates can be challenging. The bright green color of the plates makes colony visualization difficult. <br>Indeed, only fungal mycelia were visible due to a white halo resulting from quercetin degradation. However, working with liquid media allows the control of residual sample carbon source contamination through sample dilution.<br> Additionally, we were concerned agar in plates could be used as a carbon source.
-</p>
-<h3>Quercetin absorbance measurement</h3>
-<p>
-Quercetin absorbance was measured at two time points for histogram construction: at 0 days to ensure quercetin sample concentration consistent with the controls, and at 6 days to evaluate quercetin degradation (figure 5).<br> M9-quercetin and <i>Pseudomonas putida</i> K2440 samples were included as negative and positive controls, respectively.
+<center> <img src="https://static.igem.org/mediawiki/2016/2/2d/Paris_Bettancourt-4_fungi.png" alt="Assey1" style="width:600px;" align="middle"> </center><br>
 <br>
-Prior to absorbance measurement, quercetin was solubilized by diluting samples 10 fold in 0.5M NaOH, centrifuged to remove cell material, and further diluted 100X for measurement at 315 nm in a Tecan plate reader.
-<br></p>
-<h3>Quercetin degradation measurement</h3>
-<div id="figurebox">
-    <img src="https://static.igem.org/mediawiki/2016/b/b7/Paris_Bettencourt-File_Quercetin_degradation_totalstrains.jpg" alt="Quercetin degradation detail" style="width:600px; " >
-<p style="text-align: left;">
+We can see the plate becoming transparent where the mycelium have grown.
-            <b> Figure 5.</b> Kinetics of quercetin degradation by 9 promising strains. This experiment had two negative controls, one with no bacteria (black line) and one with non-quercetin degrading <i>E. coli</i>. <i>Pseudomonas putita</i> was included as a positive control. Four strains, shown in bold, degraded quercetin at a higher rate than our <i>P. putida</i> control.
-        </p>
- </div>
-<p>
-Of 186 strains tested, 50 produced quercetin levels significantly lower than controls (Figures 3 and 4). <br>20 strains degraded more than 50% of quercetin and 2 strains degraded more than 80%.<br>
-Both selective and nonselective plating methods were able to produce quercetin-degrading strains. <br>4 of the 5 strains showing the most quercetin degradation were obtained by selective plating.<br> However, 40 of the 50 strains showing significant degradation were obtained by nonselective plating.
+<h2 class="red">Week 17th-19th October</h2>
 <br>
+<br>
+We made some kinetic of the most promising strains to see the evolution of Quercetin degradation. All the culture were made in triplicate.
+<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/0/0d/Paris_Bettencourt-Cin%C3%A9tique2.jpg" alt="Kinétic of Quercetin degradation" style="width:700px;" align="middle"> </center>
+<br> As you can see, there is some variations in the absorbance that makes the curve non linear.<br>
+Here we chose not show the standard deviation to make the graph more clear.
+<br><br> To make the graph easier to understand, we chosed to normalized every daily absorbance by the control: Abs(strains)Tx/AbsTx(control). <br> Thanks to that we get an easy graph to read.<br>
+On the Y axis, we can read the % of Quercetin remaining.<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/0/02/Paris_Bettencourt-Cin%C3%A9tique2normalized.jpg" style="width:700px;" align="middle"> </center> <br>
+<br><br>
+<center> <img src="https://static.igem.org/mediawiki/2016/c/c9/Paris_Bettencourt-File_Cin%C3%A9tique.jpg" style="width:700px;" align="middle"> </center> <br>
+We sent to genome sequencing the strains FS_M75(Lysinibacillus), B2(Stenothrophomonas maltophilia), B3(Oerskovia Paurometabola), FS_M121(Microccocus Luteus). <br>
+We would like to compare, between theses species, the enzyme commonly responsible for Quercetin degradation.fimage
-</p>
-<h3>Phylogenetic analysis of quercitin degradation</h3>
-<div id="figurebox">
- <img src="https://static.igem.org/mediawiki/2016/d/dc/Paris_Bettencourt-Phylogenetic_Tree.jpg" alt="Quercetin degradation detail" style="width:950px;" >
-<p>
-<b> Figure X: </b> Phylogenetic Tree of isolated strains.</b> We constructed a phylogenetic tree of all isolated bacterial strains. Strain taxonomic classification is indicated by the color key to the left of the figure. Strains that demonstrated high quercetin degradation are marked with an asterisk. Those strains marked with a large star were selected for whole genome sequencing to look for common anthocyanin-degrading genes.
-</p>
-</div>
-<div style="clear: both">
-</div>
-<div id="figurebox">
- <img src="https://static.igem.org/mediawiki/2016/3/37/Paris_Bettencourt-Genome_Table.jpg" alt="Genome Table" style="width:600px;" >
-<p>
-<b> Figure X: Genome table.</b>
-</p>
-</div>
-<div style="clear: both">
-</div>
 <br>
-</p>
+<br>
+To finish our project, we assayed two strains FS_M75 and Pseudomonas putida with real wine stain on cotton fabric.
+<br>
+<br>
+The first experiment was made by putting a circle of cotton into a beaker with wine and then drying it by waiting a few hours.<br> We had some cells that were in a transparent media composed of M9. This M9 is composed by cells without LB (LB have been removed by centrifugation, cells were re-suspended in PBS).<br>
+And we have fabric in M9 without strains and the piece of cotton with the wine stain.<br>
-<h3> Anthocyanin Extraction and analysis </h3>
+ <margin-left> <img src="https://static.igem.org/mediawiki/2016/a/a6/Paris_Bettencourt-File_assay.jpg" alt="Assay on cotton" style="width:300px;" align="middle"> </margin-left>
-<p>
+                     <margin-right> <img src="https://static.igem.org/mediawiki/2016/5/55/Paris_Bettencourt-File_assay_on_cotton.jpg" alt="Assay on cotton" style="width:300px;" align="middle"> </margin-right>
-Anthocyanin were extracted from Vinis vitifera fruits by ethanol maceration. We confirmed the presence of anthocyanin into our extract with Lee method (Lee et Al. 2005). Thanks to our collaboration with Evry iGEM Team, we also confirmed the presence of anthocyanin by HPLC.
+<br><br>
-Pseudomonas kugensii, Stentrophomonas maltophilia and Oerskovia turbat were isolated from an M9 anthocyanin enriched media inoculate with a soil sample from Montmartre. We observed a diminution of the 525nm absorbance with TECAN, which is linked with the degradation of anthocyanin. This degradation was also observed by Evry Team with Pseudomonas putida by HPLC.
+<center> <img src="https://static.igem.org/mediawiki/2016/a/aa/Paris_Bettencourt-File_experiment_on_cotton_circle.jpg" style="width:700px;" align="middle"> </center> <br>
-</p>
+As you can see, the results are not evidential. It seems that our negative control wash the wine stain in an as efficient way as the two other strains!<br><br>
- <img src="https://static.igem.org/mediawiki/2016/thumb/0/01/Absorbancecoolopensans.png/800px-Absorbancecoolopensans.png" >
+We redo the experiment but we waited three days until the cotton was enough stained. Then we autoclaved the cotton to make sure there is no others microorganisms. We used the same experimental conditions as before.
- <img src="https://static.igem.org/mediawiki/2016/thumb/d/d2/Hplcopensans.png/712px-Hplcopensans.png" >
+<br><br>
+We made also an experiment with the platform the assay group designed. <br>
+We made three different dilution and we tested once again the ability of Pseudomonas and FS_M75 to degrade wine stain at 30°C.
 <br><br>
+<margin-left> <img src="https://static.igem.org/mediawiki/2016/e/ec/Paris_Bettencourt-File_T0_assay.jpeg" alt="Assay on cotton" style="width:300px;" align="middle"> </margin-left>
+<margin-right> <img src="https://static.igem.org/mediawiki/2016/5/53/Paris_Bettencourt-File_T6_assay.jpeg" style="width:300px;" align="middle"> </margin-right><br>
+<br>
+We used Geneious to generate consensus sequences from each bacteria isolated and sequenced, through de novo alignement between the two chromatogram files (.ab1) provided vy GATC for each sequencing. <br>
+Thus, we were able to increase the quality of our sequences, to make them more reliable. We also trimmed them when the quality was too low.<br>
+Because of bad quality, or errors in sequencing, some strains of the 187 database were deleted.<br>
+Which explain why only 173 bacteria were used to create the phylogenetic tree.<br>
+We ran the tree thanks to Geneious software with the following parameters:
+<ol> <li> The Tree build model chosen was neighbor joining. </li>
+<li> Jukes-Cantor was chosen as the genetic distance model </li>
+<li> NR_102450 , a cyanobacteria, was chosen as the outgroup </li>
+<li>We chose a cost matrix for alignment of 93% similarity (the highest proposed by Geneious)</li> </ol>
+<br>
+<b>Pay attention to put every sequences in the same direction (using the Reverse complementary option), as Geneious don't reverse them automatically ! <br> If you forget to do that, tou will have actually 2 similar trees: on assembling the forward sequences, the other one with the reverse ones.</b>
+Here is the first phylogenetic tree we were able to get. The bold strains with an * are the one that were considered as the most able to degrade quercetin, according to the previous assay. The taxonomy is summed up in the legend.<br>
+<center> <img src="https://static.igem.org/mediawiki/2016/a/a9/Paris_Bettencourt-Tree_iGEM.jpeg" alt="Phylogenetic Tree" style="width:100%;" align="middle"> </center>
-<h2 class=”red”>Methods</h2>
+<h3>A.niger kinteics</h3>
+<br>
-<h3>Anthocyanin extraction</h3>
-<p>
-<i>Vinis vitifera</i> grapes were our source for anthocyanin extraction. <br> Grapes were peeled and the skins were collected, washed and soaked overnight in ethanol with 1% HCl.<br> By trial and error, we determined that 2.5 mL of this solution per 1 g of skins was the best compromise between efficiency and the quantity of solvent used.<br> The solution was filtered with Whatman paper, with the filtrate collected, and the solvent evaporated at 37°C for several hours.<br> The dry extract was resuspended in water (10 mL for 1g of grape skin).<br>
-</p>
-<h3>Anthocyanin quantification by differential absorbance</h3>
+A.niger degradation kinetics
-<p>
+As A.niger was able to degrade all the quercetin in 48h we decided to measure precidly the degradation kinetics. We followed the quercetin degradation for 32h (almost all the quercetin was degraded<br>
-Following Lee et al. (2005), we prepared one buffer at pH 1 (0.025M potassium chloride) and a second at pH 4.5 (sodium acetate, 0.4M).<br>
+ <br>
-µL of the anthocyanin solution was mixed with 900µL of each buffers and the color was allowed to develop over 20 minutes.<br> Absorbance measurements were obtained at 510 nm and 700 nm for each solution.<br> Anthocyanin concentration was determined as a function of the four absorbance measurements, using an established formula (Lee et al., 2005).<br>
-</p>
-<h3>Protein quantification with Bradford assays</h3>
-<p>
-A stock solution of Bovine Serum Albumin (BSA) was prepared in water at 1 mg/mL.<br> 100 μL of standard dilutions of BSA solution were mixed with 1 mL of Bradford Reagent and mixed by vortexing.<br> Absorbance was measured at 595 nm. Experimental samples were treated similarly and compared to the BSA standard curve to determine concentration.<br>
-</p>
-<h3>Carbohydrate quantification with Fehling Reaction</h3>
-<p>
-µL of Fehling's A solution, 200µL of Fehling's solution B and 200µL of our carbohydrate solution into sodium acetate buffer (20µL of solution and 180 µL of buffer).<br> The Fehling reaction is measured as the loss of absorbance at 650nm relative to a blank solution without carbohydrate.<br> Quantification was achieved by comparison to a standard curve of glucose prepared at 1g/L to 5g/L.
-</p>
-<h3>Bacteria plating on selective and non-selective media</h3>
-<p>1 g of soil samples were suspended in 5 mL Phosphate Buffered Saline (PBS) then left to stand allowing large particles to settle.<br> The soil suspension was serially diluted to obtain a suitable density of microbes (typically 1:1000) then 200 µL was plated on standard Petri dishes with M9 agar with 1 g/L quercetin for selection.<br> Non-selective plating was performed on a range of rich media including FTO agar (Curry, 1976), Mossel agar (Mossel, 1967), standard LB, standard TSA and standard M9 glucose.<br>
-</p>
-<h3>Protocol for growth assay in Quercetin M9 liquid media</h3>
+<center> <img src="https://static.igem.org/mediawiki/2016/e/ee/Paris_Bettancourt-quercetin_degradation.png" alt="Phylogenetic Tree" style="width:600px;" align="middle"> </center>
-<p>Following the protocol of Dantas <i>et al.</i> 2012, all step were performed in liquid media to control soil carbon source contamination.<br> We suspended soil samples in 5 ml M9 with 1g/L quercetin at pH 7 in 50 ml Falcon tubes with 500µL of overnight culture of strains isolated from selective or non-selective plates.<br><br>
-All cultures were made in triplicate at 30°C with shaking at 150 rpm for several days. As quercetin is not soluble at pH=7, shaking important to avoid precipitation.<br></p>
-<h3> Quercetin absorbance measurement </h3>
-<p>Quercetin absorbance was measured at two time points for histogram construction: at 0 days to ensure quercetin sample concentration consistent with the controls, and at 6 days to evaluate quercetin degradation.<br> M9-quercetin and <i>Pseudomonas putida</i>K2440 samples were included as negative and positive controls, respectively.<br><br>
-Prior to absorbance measurement, Quercetin was solubilized by diluting samples 10 fold in 0.5M NaOH, centrifuged to remove cell material, and further diluted 100X for measurement at 315 nm in a Tecan plate reader.<br></p>
-<h3>PCR for 16s characterization, sequencing interpretation and phylogenetic tree construction.</h3>
-<p>To identify bacterial strains, 16S rRNA sequences were amplified through colony PCR, column purified, and Sanger sequenced by GATC.<br> The resulting sequences were submitted for BLAST comparison at ncbi.gov.<br> Alignments were performed using the Ribosomal Database Project Aligner tool (https://rdp.cme.msu.edu/),<br> and a phylogenetic tree was constructed using Geneious software with the following parameters: we used Neighbor-Joining tree building with Jukes Cantor as the genetic distance model, with a 93% similarity cost matrix for the alignment with free end gaps.<br> The tree was then exported and improved using the online Tree of Life software (http://www.tolweb.org/tree/).</p>
-<h3>PCR for genome sequencing.</h3>
+We could clearly see the exponential aspect of the curb. Almost all the quercetin degradation happened between 16h and 29h. We calculated the degreadation speed between these two moments :
+V=(C(17)-C(29))/(29-17) = 0,8g/h
-<p>We isolated bacterial DNA using the DNeasy Blood and Tissue Kit from Qiagen.<br> We submitted four strains to GATC for whole genome sequencing: NS.4 (<i>Lysinibacillus</i>), S.48 (<i>Stenothrophomonas maltophilia</i>), S.33 (<i>Oerskovia Paurometabola</i>), NS.33 (<i>Microccocus Luteus</i>) according to their sample preparation specifications.<br></p>
+This value is important to study the impact of a gene on the A.niger’s ability to degrade quercetin. If we had more time we would have done this kind of kinetics on all the fungi to compare their ability to degrade quercetin.
-<h2 class=”red”>Attributions</h2>
-<p>This project was done by Antoine Villa Antoine Poirot and Sébastien Gaultier. Anthocyanin data was obtained by Ibrahim Haouchine. <br> Thanks to our advisors Jake and Jason for all their help with the figures.<br> We would like to thank Philippe Morand from the microbiology lab of Cochin for his advice.</p>
-<img src="https://static.igem.org/mediawiki/2016/d/de/Paris_Bettencourt-sebstatic.jpeg" width="200px"/><img src="https://static.igem.org/mediawiki/2016/6/6c/Paris_Bettencourt-Antoinepstatic.jpeg" width="200px"/><img src="https://static.igem.org/mediawiki/2016/f/f6/Paris_Bettencourt-AntoineVstatic.jpeg" width="200px"/><img src="https://static.igem.org/mediawiki/2016/b/b3/Paris_Bettencourt-Ibrastatic.jpeg" width="200px"/> <br>
-<h2 class=”red”>References</h2>
-<ul>
-<li>Kanekar, P. P., Sarnaik, S. S., & Kelkar, A. S. (1998). Bioremediation of phenol by alkaliphilic bacteria isolated from alkaline lake of Lonar, India. <i>Journal of applied microbiology</i>, 85(S1).</li>
-<li>Dantas, G., Sommer, M. O., Oluwasegun, R. D., & Church, G. M. (2008). Bacteria subsisting on antibiotics. <i>Science</i>, 320(5872), 100-103.</li>
-<li>Lee, J., Durst, R. W., & Wrolstad, R. E. (2005). Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. <i>Journal of AOAC international</i>, 88(5), 1269-1278.</li>
-<li>Curry, J. C., & Borovian, G. E. (1976). Selective medium for distinguishing micrococci from staphylococci in the clinical laboratory. <i>Journal of clinical microbiology</i>, 4(5), 455.</lI>
-<li>Pillai, B. V., & Swarup, S. (2002). Elucidation of the flavonoid catabolism pathway in Pseudomonas putida PML2 by comparative metabolic profiling. <i>Applied and environmental microbiology</i>, 68(1), 143-151.</li>
-<li>Herrmann, H., Janke, D., Krejsa, S., & Kunze, I. (1987). Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H. <i>FEMS microbiology letters</i>, 43(2), 133-137.</li>
-</ul>
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