Team:Paris Bettencourt/Results
Results
Automated/DIY microplate on fabric-Image processing tool for stain measurement
An automated 96 well microplate for fabric was made using a fluorescent microplate, 96 well tip holder and laser-cut cotton circles.
- Spread a flat, wet cotton sheet on glass substrate of 96 well plate dimension.
- 96 Wet cotton circles of 6.5mm are laser cut on glass substrate.
- Remove the fabric. The circles of cotton stay on the glass substrate.
- Place the glass substrate of cotton circles on fluorescent microplate such that all circles face the wells and align well.
- Hold it tight and move the glass substrate laterally so that all the cotton circles fall into the wells.
- Put the tip plate holder on the top of the microplate and adhere it to the microplate using a glue.
- Cover it with a lid and the microplate is ready to use for experiments. The same can be done for other fabrics like silk, polystyrene, nylon etc.
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).
| COTTON | LINEN | SILK | WOOL | POLYESTER |
|---|---|---|---|---|
| GVLRYAP | STNPTSL | SILPVTR | ADIRHIK | MPRLPPA |
| RLLQYNS | MPRLPPA | MPRLPPA | YMGPSKT | MPRLPPH |
| GVKSEQL | MPRLPPA | MPRLPPA | QFDHWRN | MPRLPPA |
| WHLPAQR | MPRLPPA | AQSNPKN | MRLSVPN | MPRLPPA |
| ELAGTTW | MPRLPPA | KNANSRE | ADARYKS | MPRLPPA |
| MSNTLDP | SLLTHNM | GKNLMNM | SILPVTR | ADARYKS |
| MPRLPPA | MPRLPPA | MPRLPPA | SILPVTR | VFQTTYK |
| ISTTLFP | MPRLPPA | MPRLPPA | ADARYKS | ASSHIHH |
| TTHPRWG | MPRLPPA | MPRLPPA | PSNRQNT | GASNIWN |
| SFLVTRN | MPRLPPA | KTAMKGP | SILPVTR | GASNIWN |
| ASSHIHH | MPRLPPA | TSNRAPY | GSTSFSK | ISTTLFP |
| ALANFEP | MPRLPPA | ADARYKS | ADARYKS | GALAKDE |
| MRLSVPN | MPRLPPA | MPRLPPA | SILPVTR | MRLSVPN |
| MPRLPPA | XPRLPPA | MPRLPPA | ADARYKS | MPRLPPA |
| ERGFLLL | QPIYRVQ | ADARYKS | SILPVTR | MPRLPPA |
| SIHERAK | MPRLPPA | MPRLPPA | QFDHWRN | ADARYKS |
| VECINNC | WTNVFVG | DETCSSM | GQSVVSL | ADARYKS |
| ASSHIHH | XPRLPPX | MPRLPPA | ASSHIHH | HWNTVVS |
| HYPPVDD | MPRLPPA | MPRLPPA | MPRLPPA | METVVSS |
| ASSHIHH | XPXXPPT | XPRLPPA | ASSHIHH | MQEMRQM |
| ADARYKS | MPRLPPA | MPRLPPA | ADARYKS | SNYHWRM |
| TSDATQR | XPRLXPA | MPRLPPA | ADARYKS | NNSVSMN |
| HNWMHQN | MPRLPPX | FPSPMVG | SILPVTR | SILPVTR |
| ADARYKS | MPRLPPA | AWPYVTL | MRLSVPN | MPRLPPA |
| ADARYKS | MPRLPPA | AQSNPKN | HDSPTAA | MPRLPPA |
| TDHAHRY | ASPDQEK | KNANSRE | MPRLPPA | FRKKRKS |
| SVVMPHG | MPRLPPA | MPRLPPA | ADARYKS | TESAPTL |
| ADARYKS | QFPPPPG | MPRLPPX | ADIRHIK | SLETMSN |
| FSRSNNT | HDSPTAA | MPRLPPX | YMGPSKT | MPRLPPA |
| TDMTAPK | MPRLPPA | HDVMWQR | SILPVTR | MPRLPPA |
| MTQQLHT | MPRLPPA | MPRLPPA | ASSHIHH | MPRLPPA |
| SSHSVQR | TNLHINP | HWNTVVS | TVHVHKT | VPRLPPA |
| GLHYDHS | AGHVVPR | MLQGNGY | ADIRHIK | MPRLPPA |
| ASSHIHH | HWNTVVS | ASSHIHH | YMGPSKT | MPRLPPA |
| DPRLSPT | SILPVTR | SILPVTR | GSTSFSK | MPRLPPA |
| QGDYFTY | TLINYRG | AGHVVPR | SILPVTR | MPRLPPA |
| VTLPDPR | ADARYKS | MPRLPPA | TRPTDTI | MPRLPPA |
| VTLPDPR | MPRLPPA | MPRXPPA | GSTSFSK | MPRLPPA |
| FSRSNNT | SILPVXR | MPRLPPA | HDSPTAA | |
| ADARYKS | HDVMWQR | MPRLPPA | YMGPSKT | |
| SILPVTR |
Table 1A. Novel fabric binding domains with diverse properties.
| COTTON | LINEN | SILK | WOOL | POLYESTER |
|---|---|---|---|---|
| FSRSNNT(2) | SILPVTR(2) | AQSNPKN(2) | MRLSVPN(2) | ADARYKS(3) |
| MPRLPPA(2) | AWPYVTL(2) | ASSHIHH(3) | ||
| ASSHIHH(4) | MPRLPPA(25) | KNANSRE(2) | ADARYKS(6) | MPRLPPA(20) |
| ADARYKS(5) | SILPVTR(2) | SILPVTR(7) | ||
| MPRLPPA(20) |
Table 1B. Table of all peptide sequences observed to bind more than once. The number in parenthesis indicates the number of times the respective peptide was recovered. These candidate peptides were validated for binding affinity with an ELISA.
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 .
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.
+33 1 44 41 25 22/25
igem2016parisbettencourt@gmail.com 