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

 
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<div id=topheader> </div>
  
<h1 class="red">Microbiology Group Banner Image Goes Here </h1>
+
<!--h1 class="red">Microbiology Group: The Search for Anthocyanin Degradation in Nature </h1-->
 +
 
 +
 
 +
<!----------------------- BEGIN SUMMARY BOXES-------------------------> 
 
    
 
    
<div class="projtile1" style="margin-right:15px">
+
<div class="projtile1">
<h2 class="red">Goals</h2>
+
    <h2 class="red" style="text-align:center;">Goals</h2>
 +
    <ul>
 +
        <li>To screen soil samples from around the world for microbes that naturally degrade anthocyanin.</li>
 +
        <li>To identify enzymes linked to the most efficient anthocyanin eaters.</li>
 +
    </ul>
 +
</div>
 +
 
 +
 
 +
<div style="clear: both;"></div>
 +
 
 +
<div class="projtile3">
 +
      <h2 class="red" style="text-align:center;">Results</h2>
 +
      <ul>
 +
            <li>We collected species from all around the world through iGEM collaborations.
 +
            <li>186 bacteria were tested for degradation of quercetin.
 +
            <li>174 bacteria were identified by 16s rRNA sequencing.
 +
            <li>4 promising species were selected for whole genome sequencing.
 +
            <li>Candidate enzymes were identified from genome sequence, and passed to the enzyme group.</li>
 +
 
 +
      </ul>
 +
</div>
 +
 
 +
<div style="clear: both;"></div>
 +
 
 +
<div class="projtile4">
 +
    <h2 class="red" style="text-align:center;">Methods</h2>
 +
    <ul>
 +
        <li>Microbe cultivation
 +
        <li>Anthocyanin purification
 +
        <li>Anthocyanin & quercetin measurement
 +
        <li>16S rRNA sequencing
 +
        <li>Whole genome sequencing
 +
        <li>Phylogenetic analysis
 +
    </ul>
 +
</div>
 +
 
 +
<div style="clear: both;"></div>
 +
 
 +
<!----------------------- END SUMMARY BOXES------------------------->   
 +
 
 +
<div id=subheader>
 +
<div id="input">
 +
<h2 class="red">Abstract</h2>
 
<p>
 
<p>
Someone should write down this part
+
<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 quercetin, a compound structurally similar to anthocyanin. By phylogenetic analysis, we were able to connect quercetin 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 quercetin 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>
 
</p>
 
</p>
 +
 +
<h2 class="red"> Motivation and Background</h2>
 +
<h3>Bioprospecting and Bioremediation</h3>
 +
 +
<p>
 +
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>
 +
 +
 +
<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>Isolate pure strains and measure their activity.</li>
 +
<li>Connect the activity to specific pathways using molecular genetics.</li>
 +
</ol>
 +
</p>
 +
 +
<h3>Anthocyanidin and quercetin</h3>
 +
<p>
 +
In these experiments, we use quercetin as a chemical proxy for anthocyanins. Naturally occurring anthocyanidins are chemically diverse derivatives of a a core flavylium 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.
 +
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 quercetin, a flavonol, as a structural proxy. Quercetin 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.
 +
</p>
 +
 +
<div id="figurebox">
 +
<div style="text-align:center;">
 +
<img src="https://static.igem.org/mediawiki/2016/2/29/Paris_Bettencourt-Malvidin_and_Quercetin_chemical_structure.png" alt="Quercetin strains degradation" style="height:600px;">
 +
<p>
 +
<b>Figure 1</b> Structure and absorbance of malvidin, the most abundant anthocyanidin in wine, and quercetin, a flavonol.<p>
 +
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.<br>
 +
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.<br>
 +
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.
 +
</p>
 +
 +
</p>
 +
</div>
 
</div>
 
</div>
  
<div style="clear: both;"></div>
 
  
<div class="projtile2" style="margin-right:15px">
 
      <h2 class="red">Methods</h2>
 
      <p>
 
Strains Library
 
<br>
 
<br>
 
Natural selection on single carbon source liquid media
 
      </p>
 
 
 
    </div>
 
    <div style="clear: both;"></div>
 
  
<div class="projtile3" style="margin-right:15px">
+
 
 
<h2 class="red">Results</h2>
 
<h2 class="red">Results</h2>
<p>
+
<h3>Anthocyanin Extraction and analysis</h3>
We were able to build a library of 187 strains that were tested for their ability of Quercetin degradation.<br>
+
<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>
Theses strains come from soil sample that were collected in vignard from all arround the world.
+
<br>
+
<br>
+
In the other hand, we selected some fungi from soil sample and Quercetin single carbon source liquid media able to consumm Quercetin very efficiently.
+
  
 +
<h3>Collection of the soil samples</h3>
  
      </p>
+
<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.
  
    </div>
+
<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.
    <div style="clear: both;"></div>
+
</p>
  
<div class="projtile4" style="margin-right:15px">
+
<div id="figurebox">
    <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect" title="Detect">
+
<table border="1">
      <h2 class="red">BioBricks</h2>
+
<tr>
     
+
<th>Country</th>
    </a>
+
<th>Location</th>
      <p>
+
<th>Collector</th>
Someone should write down this part no biobricks
+
</tr>
  
 +
<tr>
 +
<td>France</td>
 +
<td>Clos Monmartre Vineyard, Paris</td>
 +
<td>Our Team</td>
 +
</tr>
  
      </p>
+
<tr>
 +
<td>France</td>
 +
<td>Cochin Port Royal, Paris</td>
 +
<td>Our team</td>
 +
</tr>
  
    </div>
+
<tr>
    <div style="clear: both;"></div>
+
<td>France</td>
 +
<td>Vaucluse region’s vineyard</td>
 +
<td>INSA-Lyon iGEM team</td>
 +
</tr>
  
<!-- boxes stuff ends here-->  
+
<tr>
 +
<td>Spain</td>
 +
<td>Barcelona</td>
 +
<td>UPF-CRG Barcelona iGEM team</td>
 +
</tr>
  
 +
<tr>
 +
<td>Spain</td>
 +
<td>Utiel Requena</td>
 +
<td>UPV Valencia iGEM team</td>
 +
</tr>
  
</style>
+
<tr>
<head>
+
<td>Australia</td>
   
+
<td>Hunter Valley</td>
</head>
+
<td>UNSW</td>
 +
</tr>
  
<div id=topheader>
+
<tr>
    </div>
+
<td>Australia</td>
    <div id=subheader>
+
<td>Sydney</td>
   
+
<td>Macquarie 2016 iGEM team</td>
    <div id="input">
+
</tr>
        <h1 class="red">Discover natural enzymes for anthocyanin degradation </h1>
+
       
+
            <h2 class="red">Highlights</h2>
+
               
+
                <h3> Goals</h3>
+
                    <ul>
+
                        <li>• To screen soil samples from around the world to find bacterial and/or fungal strains that naturally degrade anthocyanin, either as a metabolic substrate or a dedicated carbon source.</li>
+
                    </ul>
+
           
+
                <h3>Methods</h3>
+
                    <ul>
+
                        <li>Microbiome cultivation
+
                        <li>Anthocyanin extraction
+
<li>Anthocyanin/Quercitin degradation screen
+
<li>DNA extraction
+
<li>16S/18S PCR amplification and sequencing
+
<li>Whole genome sequencing
+
<li>Phylogenetic analysis
+
<li>Creation of microbial library
+
                        <li>Quantitative ELISA<p>
+
                    </ul>
+
  
                <h3>Results</h3>
+
<tr>
                    <ul>
+
<td>Namibia</td>
                        <li>Sample origin</li>
+
<td>Etosha National Park</td>
                        <li>Species isolated</li>
+
<td>Our team</td>
<li>How well samples/species degraded quercitin</li>
+
</tr>
<li>How well samples/species degraded anthocyanin</li>
+
<li>Phylogenetic tree of the different species of bacteria and fungus</li>
+
                        <li>Common candidate genes</li>
+
                    </ul>
+
                       
+
                <h3>BioBricks</h3>
+
                    <ul>
+
                        <li>BioBrick 1</li>
+
                        <li>BioBrick 2</li>
+
                        <li>BioBrick 3</li>
+
                    </ul>
+
            <h2 class="red">Abstract</h2>
+
                <p>
+
                    In this part of the project, we screened bacteria previously isolated from vineyards around the world to look for strains able to degrade flavonoids such as Quercitin and anthocyanin. This was done to find non-toxic alternatives to PERC, a toxic chemical widely used in the dry cleaning industry. Through our screen from XX locations around the world, we isolated X species capable of degrading these compounds, which were identified through 16S sequencing and subjected to phylogenetic analysis. We also chose a few promising, unknown strains of [species XX] for whole-genome sequencing to look for common enzymes. This allowed us to construct a microbial library that could be passed on to the assay team to directly test microbial enzyme activity on fabric samples.
+
               
+
<br>
+
<br>
+
In another hand, we made a selection process of microbes on a single carbon source media composed by Quercetin and soil sample. The idea was to get at the end some microbes able to use Quercetin as a carbon source. After 6 days of experiments, we had about 9 different fungi able to degrade Quercetin in a very efficient way.
+
</p>
+
  
 +
<tr>
 +
<td>Algeria</td>
 +
<td>Alger</td>
 +
<td>Our team</td>
 +
</tr>
  
            <h2 class="red"> Motivation and Background</h2>
+
<tr>
                <p>
+
<td>Croatia</td>
                  Wine stains are notoriously difficult to remove from clothing. This is true for ordinary consumers as well as for professional cleaning services, a fact that our human practices team confirmed through a widespread survey of Parisian dry cleaners. Perchloroetylene (PERC) is a common solvent used by dry cleaners to remove stains; however, it is toxic both for human health and the environment, and will be phased out of use in France by 2022. The difficulty in stain removal is due to the complex chemical composition of the wine itself, which includes phenolic compounds such as flavonoids.<br>
+
<td>Kricke</td>
 +
<td>Our team</td>
 +
</tr>
  
                    <br>Flavonoids, specifically anthocyanins, are abundant in grapes and are the main contributors to red wine pigmentation (Kennedy 2005). In order to find a more sustainable, non-toxic alternative to PERC, we screened bacteria for enzymes that break down anthocyanins, either as a metabolic substrate or as a carbon source. Microbes living in vineyard soil and on the grapes themselves have been suggested to play a role in wine quality itself (Bokulich 2016). As it seemed likely that microbes growing in vineyards would be capable of anthocyanin degradation, we focused on sample collection from a diverse range of vineyard locations. In the course of our screen, we gathered grape and soil samples from Australia, Croatia, Namibia, Spain, and France, primarily through collaboration with other iGEM teams, and tested them for flavonoid degradation.<br>
+
<tr>
 +
<td>Israel</td>
 +
<td>Jerusalem</td>
 +
<td>Our team</td>
 +
</tr>
 +
</table>
  
                    <br>We tested anthocyanins from multiple sources: one the one hand, we purchased the anthocyanin malvidin in order to make a standard curve from (Sigma?). On the other, we also isolated anthocyanin from vineyard grapes in order to have a more “natural” chemical representation sample; this had the added benefit of obtaining anthocyanins in a more cost effective manner. We also tested for degradation of the flavonolic compound quercetin, which we could purchase much more cheaply than anthocyanin.<br>
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<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;">
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<p>
 +
</p>
  
                   
+
<p>
                </p>
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<b>Figure 2</b> Location of soil samples<br>collected or obtained by the team.
 +
</div>
  
            <h2 class="red">Results</h2>
+
</div>
                           
+
  
                <h3>Selection of quercetin as an anthocyanin substitute</h3>
+
<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.
  
                    <p>
+
<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.
                        Direct testing of anthocyanin presented a challenge for the team, as anthocyanin can be difficult to isolate and purchasing large quantities is prohibitively expensive. We surveyed the literature to find an inexpensive substitute with a highly similar structure to anthocyanin. 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 (figure). Cyanindin and malvidin (most commonly found in wine) 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.  
+
 
 +
<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>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<h3>Quercetin degradation assay</h3>
 +
 +
<p>
 +
Each of the 189 isolated strains was innoculated into M9 quercitin medium in triplicate and incubated at 37 C for 6 days. At the end of the period, quercitin concentration was measured by absorbance.<br><br>
 +
 +
 
 +
Of 186 strains tested, 50 produced quercetin levels significantly lower than controls (Figures 3 and 4). 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. 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.
 +
<br><br>
 +
 
 +
 
 +
<br></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</b> Quercetin degradation by 186 microbes collected from global soil samples. Single colonies were inoculated in M9 minimal medium and grown for 6 days. Quercetin was measured by absorbance. Strains to the extreme left of the figure represent the highest-degrading strains. Red bars represent strains isolated from selective media and blue from non selective media.
 
<br>
 
<br>
<br>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 (in4?). 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, leading to the possibility that quercetin degradation could impact anthocyanin stability (need more background here?).
 
 
<br>
 
<br>
<br>Concurrent to the quercetin degradation screen, we optimized an anthocyanin isolation protocol from grapes in order to similarly test our samples on anthocyanin.
 
                    </p>
 
                <h3>Soil sample collection map</h3>
 
                    <p>
 
                        Our sample collection included soil and grapes from XX locations in France, XX in Europe including X, and X locations in Australia (more?). We tested soil resuspensions, individual isolated microbes, and whole cell extracts for their ability to degrade quercetin and anthocyanin. Samples from locations in XX were capable of degrading q or a (X for q, Y for a), and samples from YY could degrade both (figure).
 
  
 +
</p>
 +
</div>
 +
</div>
  
<center> <img src="https://static.igem.org/mediawiki/2016/1/1f/Paris_Bettencourt-File_Quercetin_.jpg" alt="Quercetin strains degradation" style="width:900px;" align="middle"> </center>
+
<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:900px; " >
 +
 
 +
<p style="text-align: left;">
 +
            <b> Figure 4</b> Quercetin degradation detail. The top-performing strains included those isolated from both selective and nonselective media. Strains marked with an asterisk were selected for further investigation. Red bars represent strains isolated from selective media and blue from non selective media.
 +
        </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.
 +
<br><br>8 top-performing strains were selected for detailed valication in a time-course assay, and all were able to degrade quercitin significantly better than E. coli negative controls. <br>We observed a 50% degradation over 6 days in the best case.</p>
 +
 
 +
<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:450px; " >
 +
 
 +
<p style="text-align: left;">
 +
<div style="text-align:center;">
 +
            <b> Figure 5</b> Kinetics of quercetin degradation by 8 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>
 +
</div>
 +
 
 +
 
 +
<h3>Phylogenetic analysis of quercetin degradation</h3>
 +
<p>
 +
Using the 16s rRNA sequences, we constructed a global phylogeny of all the assayed strains (Figure 6). <br>Quercitin-degrading activity was concentrated among the Pseudomonas genus, where most of the strains were positive.<br> However many diverse species were able to significantly degrade quercitin, suggesting that the phenotype may be highly variable in evolution.<br>
 +
 +
 
 +
</p>
 +
<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 6: 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>
 +
 
 +
 
 +
<h3>Mining genomes for quercetin-degrading enzymes</h3>
 +
 
 +
<p>
 +
 
 +
Four of our top-degrading strains, representing diverse phylogentic lineages, were selected for whole genome sequencing. <br>As of the wiki freeze, the genome sequences were not complete. So, as a proxy, we obtained complete genome sequences of their nearest relatives in GenBank.<br> BLAST searches against the genomes revealed the presence of several candidate quercitin-degrading enzymes, including laccase and XylE.
 +
<br>
 
<br>
 
<br>
                    </p>
+
The results of these genomic analysis were passed to the enzyme group, where these enzymes form the basis of our synthetic stain fighting product.
 +
</p>
 +
<div id="figurebox">
 +
<img src="https://static.igem.org/mediawiki/2016/3/37/Paris_Bettencourt-Genome_Table.jpg" alt="Genome Table" style="width:900px;" >
 +
<p>
 +
<b> Figure 7: Genome table.</b> Genome sequences were analyzed for the presence of potential anthocyanin degrading enzymes as identified in the Enzymes Project. The genes of at least one of the six candidates were identified for each of the sequenced strains. The number of + signs indicated represent the number of gene copies in the bacterial genome.
 +
</b>
 +
</p>
 +
</div>
 +
<div style="clear: both">
 +
</div>  
  
            <h2 class="red">Methods</h2>
 
  
                <h3>Anthocyanin isolation protocol</h3>
 
                           
 
                    <p>
 
                        blabla ###.
 
                    </p>
 
  
                <h3>Preparation of fabric samples for panning</h3>
+
<br>
                       
+
</p>
                    <p>
+
                        Fabrics were washed with x prior to panning to remove coatings, preservatives or other treatments that may have been applied in the factory.
+
                    </p>
+
  
                <h3>Detailed protocol for phage display</h3>
 
                    <p>
 
                        10 µl of phage was mixed with 1 gram of fabric...<br>
 
                    <br>Sequence similarity was calculated as BLOSUM. Trees were made using Geneious with nearest neighbor joining.
 
                    </p>
 
  
 +
<br><br>
  
                <h3>Sequence clustering analysis</h3>
 
  
                <h3>Binding quantification with ELISA</h3>
 
<!--                    <p>Blablabla fill this in...</p>
 
-->
 
  
            <h2 class="red">Attributions</h3>
+
<h2 class=”red”>Methods</h2>
                <p>
+
                    This project was done mostly by Antoine Villa Antoine Poirot and Sébastien Gaultier. Put here everyone who helped including other iGEM teams
+
<h3>Anthocyanin extraction</h3>
                </p>
+
<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>
  
            <h2 class="red">References</h2>
+
<h3>Anthocyanin quantification by differential absorbance</h3>
                <ul>
+
<p>
                    <li>Sweetlove, L. J., & Fernie, A. R. (2013). The Spatial Organization of Metabolism Within the Plant Cell. Annual Review of Plant Biology, 64(1), 723–746.</li>
+
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>
 +
100 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>
 +
200µL 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>
  
                    <li>Lee, H., DeLoache, W. C., & Dueber, J. E. (2012). Spatial organization of enzymes for metabolic engineering. Metabolic Engineering, 14(3), 242–251.</li>
+
<h3>Protocol for growth assay in Quercetin M9 liquid media</h3>
 +
<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>
  
                    <li>Pröschel, M., Detsch, R., Boccaccini, A. R., & Sonnewald, U. (2015). Engineering of Metabolic Pathways by Artificial Enzyme Channels. Frontiers in Bioengineering and Biotechnology, 3(Pt 5), 123–13.</li>
+
<h3>PCR for genome sequencing.</h3>
                </ul>
+
  
</div>
+
<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>
</div>
+
 
 +
<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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    Assay
 
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Latest revision as of 03:51, 20 October 2016


Goals

  • To screen soil samples from around the world for microbes that naturally degrade anthocyanin.
  • To identify enzymes linked to the most efficient anthocyanin eaters.

Results

  • We collected species from all around the world through iGEM collaborations.
  • 186 bacteria were tested for degradation of quercetin.
  • 174 bacteria were identified by 16s rRNA sequencing.
  • 4 promising species were selected for whole genome sequencing.
  • Candidate enzymes were identified from genome sequence, and passed to the enzyme group.

Methods

  • Microbe cultivation
  • Anthocyanin purification
  • Anthocyanin & quercetin measurement
  • 16S rRNA sequencing
  • Whole genome sequencing
  • Phylogenetic analysis

Abstract

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 quercetin, a compound structurally similar to anthocyanin. By phylogenetic analysis, we were able to connect quercetin degradation to specific bacterial phyla and genus including Micrococcus, Pseudomonas, Lysinibacillus and Oerskovia. The most effective strains were further characterized by whole genome sequencing to identify enzymes linked to natural quercetin 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.

Motivation and Background

Bioprospecting and Bioremediation

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.


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 :

  1. Select organisms from a contaminated environment, where enzymatic decontamination may have naturally evolved.
  2. Isolate pure strains and measure their activity.
  3. Connect the activity to specific pathways using molecular genetics.

Anthocyanidin and quercetin

In these experiments, we use quercetin as a chemical proxy for anthocyanins. Naturally occurring anthocyanidins are chemically diverse derivatives of a a core flavylium 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. 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 quercetin, a flavonol, as a structural proxy. Quercetin 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.

Quercetin strains degradation

Figure 1 Structure and absorbance of malvidin, the most abundant anthocyanidin in wine, and quercetin, a flavonol.

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.

Results

Anthocyanin Extraction and analysis

Anthocyanins were extracted from Vinis vitifera 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.

Collection of the soil samples

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 Facture Proforma, printed out by the sample donor and included in the shipment.
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.

Country Location Collector
France Clos Monmartre Vineyard, Paris Our Team
France Cochin Port Royal, Paris Our team
France Vaucluse region’s vineyard INSA-Lyon iGEM team
Spain Barcelona UPF-CRG Barcelona iGEM team
Spain Utiel Requena UPV Valencia iGEM team
Australia Hunter Valley UNSW
Australia Sydney Macquarie 2016 iGEM team
Namibia Etosha National Park Our team
Algeria Alger Our team
Croatia Kricke Our team
Israel Jerusalem Our team
world_Microbiome

Figure 2 Location of soil samples
collected or obtained by the team.

Preparation of the microbe library

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.
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.
The resulting culture was incubated at 30 C for 48 hours, then re-streaked to eliminate potential contamination.
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.

Quercetin degradation assay

Each of the 189 isolated strains was innoculated into M9 quercitin medium in triplicate and incubated at 37 C for 6 days. At the end of the period, quercitin concentration was measured by absorbance.

Of 186 strains tested, 50 produced quercetin levels significantly lower than controls (Figures 3 and 4). 20 strains degraded more than 50% of quercetin and 2 strains degraded more than 80%.
Both selective and nonselective plating methods were able to produce quercetin-degrading strains. 4 of the 5 strains showing the most quercetin degradation were obtained by selective plating.
However, 40 of the 50 strains showing significant degradation were obtained by nonselective plating.


Quercetin strains degradation

Figure 3 Quercetin degradation by 186 microbes collected from global soil samples. Single colonies were inoculated in M9 minimal medium and grown for 6 days. Quercetin was measured by absorbance. Strains to the extreme left of the figure represent the highest-degrading strains. Red bars represent strains isolated from selective media and blue from non selective media.

Quercetin degradation detail

Figure 4 Quercetin degradation detail. The top-performing strains included those isolated from both selective and nonselective media. Strains marked with an asterisk were selected for further investigation. Red bars represent strains isolated from selective media and blue from non selective media.

Working with M9 quercetin plates can be challenging. The bright green color of the plates makes colony visualization difficult.
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.
Additionally, we were concerned agar in plates could be used as a carbon source.

8 top-performing strains were selected for detailed valication in a time-course assay, and all were able to degrade quercitin significantly better than E. coli negative controls.
We observed a 50% degradation over 6 days in the best case.

Quercetin degradation detail

Figure 5 Kinetics of quercetin degradation by 8 promising strains. This experiment had two negative controls, one with no bacteria (black line) and one with non-quercetin degrading E. coli. Pseudomonas putita was included as a positive control. Four strains, shown in bold, degraded quercetin at a higher rate than our P. putida control.

Phylogenetic analysis of quercetin degradation

Using the 16s rRNA sequences, we constructed a global phylogeny of all the assayed strains (Figure 6).
Quercitin-degrading activity was concentrated among the Pseudomonas genus, where most of the strains were positive.
However many diverse species were able to significantly degrade quercitin, suggesting that the phenotype may be highly variable in evolution.

Quercetin degradation detail

Figure 6: Phylogenetic Tree of isolated strains. 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.

Mining genomes for quercetin-degrading enzymes

Four of our top-degrading strains, representing diverse phylogentic lineages, were selected for whole genome sequencing.
As of the wiki freeze, the genome sequences were not complete. So, as a proxy, we obtained complete genome sequences of their nearest relatives in GenBank.
BLAST searches against the genomes revealed the presence of several candidate quercitin-degrading enzymes, including laccase and XylE.

The results of these genomic analysis were passed to the enzyme group, where these enzymes form the basis of our synthetic stain fighting product.

Genome Table

Figure 7: Genome table. Genome sequences were analyzed for the presence of potential anthocyanin degrading enzymes as identified in the Enzymes Project. The genes of at least one of the six candidates were identified for each of the sequenced strains. The number of + signs indicated represent the number of gene copies in the bacterial genome.




Methods

Anthocyanin extraction

Vinis vitifera grapes were our source for anthocyanin extraction.
Grapes were peeled and the skins were collected, washed and soaked overnight in ethanol with 1% HCl.
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.
The solution was filtered with Whatman paper, with the filtrate collected, and the solvent evaporated at 37°C for several hours.
The dry extract was resuspended in water (10 mL for 1g of grape skin).

Anthocyanin quantification by differential absorbance

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).
100 µL of the anthocyanin solution was mixed with 900µL of each buffers and the color was allowed to develop over 20 minutes.
Absorbance measurements were obtained at 510 nm and 700 nm for each solution.
Anthocyanin concentration was determined as a function of the four absorbance measurements, using an established formula (Lee et al., 2005).

Protein quantification with Bradford assays

A stock solution of Bovine Serum Albumin (BSA) was prepared in water at 1 mg/mL.
100 μL of standard dilutions of BSA solution were mixed with 1 mL of Bradford Reagent and mixed by vortexing.
Absorbance was measured at 595 nm. Experimental samples were treated similarly and compared to the BSA standard curve to determine concentration.

Carbohydrate quantification with Fehling Reaction

200µL 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).
The Fehling reaction is measured as the loss of absorbance at 650nm relative to a blank solution without carbohydrate.
Quantification was achieved by comparison to a standard curve of glucose prepared at 1g/L to 5g/L.

Bacteria plating on selective and non-selective media

1 g of soil samples were suspended in 5 mL Phosphate Buffered Saline (PBS) then left to stand allowing large particles to settle.
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.
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.

Protocol for growth assay in Quercetin M9 liquid media

Following the protocol of Dantas et al. 2012, all step were performed in liquid media to control soil carbon source contamination.
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.

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.

Quercetin absorbance measurement

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.
M9-quercetin and Pseudomonas putidaK2440 samples were included as negative and positive controls, respectively.

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.

PCR for 16s characterization, sequencing interpretation and phylogenetic tree construction.

To identify bacterial strains, 16S rRNA sequences were amplified through colony PCR, column purified, and Sanger sequenced by GATC.
The resulting sequences were submitted for BLAST comparison at ncbi.gov.
Alignments were performed using the Ribosomal Database Project Aligner tool (https://rdp.cme.msu.edu/),
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.
The tree was then exported and improved using the online Tree of Life software (http://www.tolweb.org/tree/).

PCR for genome sequencing.

We isolated bacterial DNA using the DNeasy Blood and Tissue Kit from Qiagen.
We submitted four strains to GATC for whole genome sequencing: NS.4 (Lysinibacillus), S.48 (Stenothrophomonas maltophilia), S.33 (Oerskovia Paurometabola), NS.33 (Microccocus Luteus) according to their sample preparation specifications.

Attributions

This project was done by Antoine Villa Antoine Poirot and Sébastien Gaultier. Anthocyanin data was obtained by Ibrahim Haouchine.
Thanks to our advisors Jake and Jason for all their help with the figures.
We would like to thank Philippe Morand from the microbiology lab of Cochin for his advice.


References

  • Kanekar, P. P., Sarnaik, S. S., & Kelkar, A. S. (1998). Bioremediation of phenol by alkaliphilic bacteria isolated from alkaline lake of Lonar, India. Journal of applied microbiology, 85(S1).
  • Dantas, G., Sommer, M. O., Oluwasegun, R. D., & Church, G. M. (2008). Bacteria subsisting on antibiotics. Science, 320(5872), 100-103.
  • 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. Journal of AOAC international, 88(5), 1269-1278.
  • Curry, J. C., & Borovian, G. E. (1976). Selective medium for distinguishing micrococci from staphylococci in the clinical laboratory. Journal of clinical microbiology, 4(5), 455.
  • Pillai, B. V., & Swarup, S. (2002). Elucidation of the flavonoid catabolism pathway in Pseudomonas putida PML2 by comparative metabolic profiling. Applied and environmental microbiology, 68(1), 143-151.
  • Herrmann, H., Janke, D., Krejsa, S., & Kunze, I. (1987). Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H. FEMS microbiology letters, 43(2), 133-137.


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