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

Introduction

The microbiology team aims to identify wine stain degrading microorganisms.

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

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

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

This bacteria identification was based on two different approaches:

• We 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 colour degradation.

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 restreak and isolation, determine an efficient way to characterize the strains.
• For the second approach, where we test directly an anthocyanin-degradation natural metabolism, define the controls.

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


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

• a chroman ring
• an additional aromatic ring on C2

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

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

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

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

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

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

Week 11th - 17th July

Week 17th - 25th July

Church paper

We found a paper 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 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.

This protocol have some issues.

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

As we found in several papers, the regular technique to isolate microorganisms from the soil is to dilute the sample and directly plate on agar. A dilution of 10^(-1) correspond to a diltion 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. To remove the carbon contamination from the soil he filtered the sample with a 0.22µL filter. Then he cultivated the filter in a liquid medium before plating. We decided to test this protocol.

Screening assay design

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 decidede to plate 100 μL of medium just after inoculation and 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 on that medium this 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 grow on it it means that there was 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 meidum were we want to isolate the bacteria.
-M9 glucsose quercetine (M9 GQ) : this medium is necessary to test if quercetin is toxic form microorganisms, if we have less growth than in M9 glucose that means that quercetin is toxic.

As it is done in church's protocol the incubation time between each cultivation step is 7 days.
In parallel we decided to test the filtration of the soil samples. In this part of the assay samples would be filtered, one inoculated in a M9 medium and the other in a M9 quercetin medium.

Week 25th - 31th July

Quercetin medium preparation

As soon as the quercetin arrived, we decided to start the assay. However it was found that unlike anthocyans, quercetin was mostly insoluble in water. this causes problems for medium preparation will the organisms be able to degrade an insoluble molecule in a liquid broth ? But it as also a problem for quercitin quantification, we thought that we could measure the degradation of quercetin with the absorbance, however if quercetin is not soluble in water it is impossible to measure this absorbance.

We tried to solubilize querctin 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 quercitin, so an absorbance assay was impossible. However, even at 1g/L the liquid was a little yellow, so quercitin was at least slightly soluble. We thought that the little quantity of soluble quercitin 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.

Week 01th - 7th August

Result of the assay after two days

Growth result in plates :
- The control plates with M9 showed growth until 10^(-3), that means that if we want to avoid carbon contamination in plate 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), that means that quercitin is toxic for some organisms. - The plates with M9 QG showed growth until a dilution of 10^(-2) because of the carbon contamination.
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 7th-15th August

Separation in two project

At this point we divided the project in two approaches:
-The goal of the first approach (approach 1) is making a huge library of strains that were isolated from soil sample.
We would like, from a given soil sample, to isolate as much as we can of bacteria diversity.
Then we could test each strain on Quercetin liquid media and report Quercetin degradation.
Thanks to our collaboration with other iGEM team, we have sample from some place all around the world.
We would like to compare the diversity of bacteria between each soil sample taken from all around the world from vignard for instance.

At the end we will have a library with some strains that are able to degrade Quercetin and some other 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.

To achieve that, here are the step of the protocol for approach 1:

We plate at 10^-3 dilution (we choose this dilution thanks to previous experiments) to the soil sample on a non-selective agar media (LB, TSA, M9Glucose) but also on selective media (FTO for micrococcus, Agar from Mossel for Bacillus Cereus, M9 Quercetin).
We wait one or two days at 30°C.
When we have some colonies, we restrike all of them on LB plate in order to isolate a maximum of different strains. Once the restriked bacteria grew enough, we give them a name to store them in the library.

FS_M69 stand for Frank&Stain_Microbesgroup69

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…



Then we make an overnight culture in Tryptic Soy Broth and systematically we characterise our strain with a 16sPCR from colonies restriked on LB plates.
When the overnight culture is over, we inoculate 100ul of this directly in a 5ml M9 1g/L Quercetin falcon tube in triplicate. We also make two glycerol stocks. One at -80°C the other at -20°C (to be reused more easily).
For the assay on liquid media Quercetin, we are quantifying Quercetin degradation.
We are able to see Quercetin degradation thanks to the absorbance. Quercetin in solution is not soluble at least just a little. So we cannot detect absorbance at a pH=7.



But, when we put our solution at pH=12, we are able to see a nice absorbance spectrum with a pic of absorption around 315nm.
With a dilution a hundred times in NaOH we are able to have a good absorbance value for 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.



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 as a positive control.
We made two sample: 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 main the three values. And we are able to calculate the standard deviation.
The value in y axis are a percentage of remaining Quercetin.



As you can see, none of the strains are able to degrade Quercetin except or positive control.
The problem 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 as a dilution factor. The problem is that we are not able to see on Quercetin plate colonies when we restrike them to make sure they are pure.
Because of the green color, 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. Theses strains won’t be selected with this technic using a single source carbon. But these strains are maybe able to degrade Quercetin as a toxic compound using cytochrome p etc.

So for the next assay we choose to higher 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 bacteria diversity for the database.



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 theses 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.

-The goal of the second approach (approach 2) is to select directly from samples microbes that are able to degrade Quercetin.
In this approach we are only selecting strains degrading Quercetin.
The idea is to inoculate in our singe carbon source directly some soil sample diluted at different range.