Difference between revisions of "Team:Paris Bettencourt/Results"

Selected Peptides from Binding Domain Group

Phage display yields peptides binding cotton, linen, wool, polyester and silk.
The phage library was panned against fabric samples of cotton, linen, wool, polyester and silk. The cycle of phage binding, elution, and amplification was repeated three times for each fabric. Following enrichment, individual clones were isolated and sequenced to determine the sequence of their displayed peptide. We isolated and sequenced at least 40 phage plaques from each fabric.

Microbes that degrade indigo

We succeeded in finding 16 microbes that grow on denim, covered with M9 media.
After two rounds of culture in liquid media with indigo and measurements of absorbance as a way to assess indigo degradation, we found 3 strains highly capable at degrading indigo : Streptmoyces fumigatiscleroticus , Streptomyces coelicolor and Pantoea agglomerans .

Bpul laccase degrades indigo

Structural formulas of two substrates (indigo carmine, and indigo) of Bpul laccase are showing structural similarity. Since it has been shown that bpul degrades indigo carmin, we went to prove its ability to degrade indigo. All results shown in the figure above are made with our bpul gene expressed in the E. coli strain BL21(DE3). The protein gel (figure above B) is showing an over-expression of the bpul, after the induction with IPTG. When comparing uninduced bpul sample with the control sample (BL21(DE3)) we can notice leakiness of our promoter, which is probably due to great transcriptional strength of T7 polymerase. In the degradation experiments (figure above C and D), both the transformant BL21(DE3)-bpul, and the negative control BL21(DE3) have been induced with IPTG. The cell extracts of both strains were tested for the ability to degrade indigo carmine and indigo. 10µM acetosyringone (ACS) was added to the reaction mixture to facilitate the oxidation of the substrates. ACS is a known mediator of the laccases and it is often used and needed in the studies of the laccase activitiy As expected, the 1mM indigo carmine solution is entirely degraded within the 13 hours of the experiment in the cell extract which contains overexpressed bpul. Additionally, we have shown that more than 60% of indigo can be degraded by bpul laccase in the 13 hours of the experiment. It is important to notice, that in longer experiments would probably lead to even more degradation since the degradation curve didn't reach the saturation.

Construction of the strain database by the microbiology team

This summer, our group designed an experiment to find the best stain fighting enzymes that nature has to offer. We build a big database with 186 strains through selective and non-selective plating on different media.

• We managed to isolate species from all around the world through iGEM collaborations.
• 186 bacteria were tested for quercetin degradation.
• A 186 bacterial database was built and characterized.
• 20 strains produced more than 50% quercitin degradation.
• 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.

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Assay

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Microbiology

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Enzyme

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Binding

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Indigo

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Model