Difference between revisions of "Team:Paris Bettencourt/Results"
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An automated 96 well microplate for fabric was made using a fluorescent microplate, 96 well tip holder and laser-cut cotton circles. | An automated 96 well microplate for fabric was made using a fluorescent microplate, 96 well tip holder and laser-cut cotton circles. | ||
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| − | < | + | <li> Spread a flat, wet cotton sheet on glass substrate of 96 well plate dimension. |
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| − | < | + | <li> 96 Wet cotton circles of 6.5mm are laser cut on glass substrate. |
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| − | < | + | <li> Remove the fabric. The circles of cotton stay on the glass substrate. |
| − | + | ||
| − | < | + | <li> Place the glass substrate of cotton circles on fluorescent microplate such that all circles face the wells and align well. |
| − | + | ||
| − | < | + | <li> Hold it tight and move the glass substrate laterally so that all the cotton circles fall into the wells. |
| − | + | ||
| − | < | + | <li> Put the tip plate holder on the top of the microplate and adhere it to the microplate using a glue. |
| − | + | ||
| − | < | + | <li> 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. |
| + | |||
| + | </ul> | ||
An Image processing plugin was developed to measure the pixel intensity of each well of the scanned image of Microplate on fabric. The relative change in pixel intensity is the quantitative measure for degradation of stain. | An Image processing plugin was developed to measure the pixel intensity of each well of the scanned image of Microplate on fabric. The relative change in pixel intensity is the quantitative measure for degradation of stain. | ||
Revision as of 00:40, 20 October 2016
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
From approximately 20 sequences for 5 different fabrics each we picked total of 9 peptide sequence to pass on to Enzyme search group to fuse it with enzymes they are working on and study their efficiency in binding fabrics.
The peptides are named FBD 1-9 for Fabric binding domain - 1 to 9.
Out of the 9 sequences 5 are specific, i.e, one sequence specific for each fabric. The sequences were not selected based on the consensus sequence but their repeatition in that particular fabric or some unique feature in the peptide. and the other 4 sequences are non specific, found in multiple fabrics.
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FBD1 - Is non specific, it is repeated in all 5 fabrics.
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FBD 2, 3, 4- Repeated in all fabric.
FBD2-FBD3-
FBD4-
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FBD5 - Specific to Wool, it was repeated 3 times out of 16 sequences.
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FBD6 - Specific to cotton
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FBD7 - Specific to Silk
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FBD8 - Specific to Linen, the sequence was selected because it has multiple proline residues
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FBD9 - Specific to polyester, it was selected because of its high number of positively charged amino acids
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FBD10 - Picked from literature, Guo et.al, (2013) it will serve as a positive control - shown to bind to cellulose
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FDB11 - Random amino acids put together(7aa) to be used as negative control.
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.
+33 1 44 41 25 22/25
igem2016parisbettencourt@gmail.com 