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.
Our phage display experiments were performed with the commercial Ph.D.-7 Phage Display Peptide Library (NEB # E8100S). This library consists 100 copies each of 109 phages expressing a randomized 7-mer peptide fused to the phage N-terminal pIII coat protein. The displayed peptide is separated by a short Gly-Gly-Gly-Ser linker to minimize interactions between the displayed peptide and the phage itself.

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 (Table 1).

We focused our analysis on phage sequences that appeared at least twice. Isolated peptides displayed a range of fabric specificities, sequence motifs and phyicochemical properties (Table 2). Nine peptide sequences were selected for further analysis, chosen with a preference for chemical diversity and highly enriched peptides that appeared in many clones. Five of the nine peptides were fabric-specific, meaning they were isolated from only one fabric, while the others were found to bind multiple fabrics. The peptides were designated FBD 1-9 (Table 3).

Validation of the peptides using ELISA

The nine selected FBD peptides were validated with ELISA. Briefly, a clonal library of phage expressing each peptide was incubated with each of five fabric samples under conditions similar to the original panning experient. Following extensive washing, the fabric was incubated with an anti-phage primary antibody, then a secondary antibody linked to HRP, the Horse Radish Peroxidaze. The HRP enzyme produces a pigmented product, allowing semi-quantitative measurement of the level of fabric-bound phage.

In figure 1, we compare the ELISA signal before and after washing for phage displaying each of the FBD peptides against each fabric. In most cases, the phage are retained after washing and produce an ELISA signal significantly above control. Peptides were found to have a range of affinities for different fabrics. Notably wool appeared to be difficult to bind, with only FBD9 staying bound after washing.

Repeated washing reduced signal, allowing a quantitative measurement of binding affinitiy. In a typical case of FBD9 binding to cotton, more than 50% signal was retained after 10 rounds of washing. Analysis of these data is ongoing, with the goal of estimating the KD for each binding interaction.

Figure 1 Fabric binding domain validation by ELISA. A. ELISA for each FBD incubated with each fabric after two rounds of washing, normalized to the level after a single round. B. Control matrix repeating the procedure of panel A without phage. C. ELISA signal determined ater multple rounds of washing. Points represent individual measurements from three replicates. Lines represent best-fit to an exponential decay model.

Catechol dioxydases were expressed with and without Fabric Binding Domains and their functionality was demonstrated in both cases

Six candidate plant and bacterial enzymes were tested for expression in E. coli
Three out of six enzymes were functionally expressed in E. coli and tested for their ability to degrade their natural substrate
Feasibility of fusing proteins with FBDs was tested by fusing them to GFP and assessing their GFP ability and their ability to bind to several fabrics
The selected enzymes were fused with Fabric Binding Domains and their activity was still observed

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 .

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