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Revision as of 21:16, 19 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 managed to isolate species from all around the world through iGEM collaborations.
- 189 bacteria were tested for quercetin degradation.
- 10 fungi were tested for quercitin degradation.
- A 178 bacterial database was built and characterized.
- A phylogenetic tree of 174 different bacterial species was created .
- 3 bacterias were shown able to degrade anthocyanin
- Common candidate genes were selected through genome sequencing of 4 different bacterial strains.
Methods
- Microbiota cultivation
- Anthocyanin purification
- Anthocyanin & Quercitin 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 182 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 Micrococcus, Pseudomonas, Lysinibacillus and Oerskovia. 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.
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.
Team | Year | Project |
---|---|---|
Leicester | 2012 | Polystyrene Biodegradation |
UCL | 2012 | Plastic Republic |
TU-Munich | 2013 | PhyscoFilter |
IIT Delhi | 2014 | Oxide Decontamination |
NEFU China | 2014 | Cadmium Decontamination |
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 anthcyanin could be revealed with a classic bioremediation strategy :
- Select organisms from a contaminated environment, where enzymatic decontamination may have naturally evolved.
- Isolate pure strains and measure their activity.
- Connect the activity to specific pathways using molecular genetics.
Anthocyanidin and Quercitin
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. 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.
![Quercetin strains degradation](https://static.igem.org/mediawiki/2016/3/3c/Quercetin_structure.png)
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Figure X: Structure and absorbance of malvidin, an anthocyanidin, 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 (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, 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
Anthocyanin were extracted from Vinis vitifera fruits. It skin was separated from the rest of the fruit and macerated overnight into an ethanol solution with 1% of chloridric acid. After the maceration, the obtained solution was filtrated with a paper filter to eliminate the solid particle and evaporated at 37°C at 150 rpm. We confirmed the presence of anthocyanin with HPLC, colour variation with pH and HPLC.
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 | Macquarie 2016 iGEM team | |
Namibia | Etosha National Park | Our team |
Algeria | Alger | Our team |
Croatia | Kricke | Our team |
Israel | Jerusalem | Our team |
![world_Microbiome](https://static.igem.org/mediawiki/2016/f/f8/Paris_Bettencourt-File_Sample_World_microbio.jpg)
Location of soil samples collected or obtained by the team.
Preparation of the microbe library
For safety, 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. In nonselective plating, soil eluate was serially diluted and plated on rich media (Trypic Soy Agar, TSA) or minimal media (M9 Glucose). In selective plating, soil eluate was used to innoculate a 20 mL culture of M9 quercitin media, in which quercitin was the only supplied carbon source. The resulting culture was incubated at 37 C for 48 hours, then serially diluted and plated on TSA. The purpose of selective plating was to enrich for microbes with the ability to metabolize quercitin.
In each case, single colonies were isolated then re-streaked to eliminate potential contamination. To maximize the library diversity, we preferentially chose colonies with unique morphology and we took no more than 5 clones from a single soil sample.
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.
Quanitification of quercitin degradation
Single colonies from the selective and nonselective microbe libraries were innoculated in M9 Glucose media containing X g of quercitin. Samples were cultured at 37 C for six days. Following incubaction, samples were centrifuged to remove cells and contaminants. Quercitin was measured by absorbance at 375 nm.
Of 182 strains tested, 50 produced quercitin levels significantly lower than controls (Figure X). 20 strain produced more than 50% quercitin degradation and 2 strains degraded quercitin more than 80%.
Both selective an nonselective plating methods were able to produce quercitin-degrading strains. 4 of the 5 strains showing the most quercitin degradation were obtained by selective plating. However, 40 of the 50 strains showing significant degradation were obtained by nonselective plating.
Phylogenetic analysis of quercitin degradation
Testing microbes with real wine and real fabrics
Mining genomes for quercitin-degrading enzymes
![Quercetin strains degradation](https://static.igem.org/mediawiki/2016/1/1f/Paris_Bettencourt-File_Quercetin_.jpg)
Figure X: Quercitin degradation by 189 microbes collected from global soil samples Single colonies were inoculated in M9 minimal medium and grown for 6 days. Quercitin was measured by absorbance.
![Quercetin degradation detail](https://static.igem.org/mediawiki/2016/3/34/Paris_Bettencourt-File_Quercitin_Detail.jpeg)
Figure X: Quercitin degradation detail The top-performing strains included isolated from both selective and nonselective media. Strains marked with an asterisk were selected for further investigation.
![Quercetin degradation detail](https://static.igem.org/mediawiki/2016/d/dc/Paris_Bettencourt-Phylogenetic_Tree.jpg)
Figure X: Phylogenetic Tree.
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.
![](https://static.igem.org/mediawiki/2016/d/de/Paris_Bettencourt-sebstatic.jpeg)
![](https://static.igem.org/mediawiki/2016/6/6c/Paris_Bettencourt-Antoinepstatic.jpeg)
![](https://static.igem.org/mediawiki/2016/f/f6/Paris_Bettencourt-AntoineVstatic.jpeg)
![](https://static.igem.org/mediawiki/2016/b/b3/Paris_Bettencourt-Ibrastatic.jpeg)
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.