Composite

Part:BBa_K1321357:Experience

Designed by: Chris N Micklem   Group: iGEM14_Imperial   (2014-10-08)

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1321357

Edinburgh 2015 iGEM team
We improved and expanded characterisation on the part BBa_K1321357, a cellulose binding domain (CBD) submitted by the Imperial College 2014 team. After confirming expression through SDS PAGE we incubated iso-standard paper chads from Whatman 54 filter paper in a crude cell lysate containing the CBD bound to super folded GFP for ten minutes. We then let the protein saturated chads sit in a large volume of 1 x PBS buffer at pH 7.4 (to remain consistent with Imperial’s initial characterisation). At different time points chads were taken from the buffer and placed into a 96 well plate, over 30 minutes. The decrease in fluorescence over time was indicative of relative binding affinity, to Whatman 54 paper. The graph below shows our results.
Dissociation curve of sfGFP-CBDcex.jpg


Paris Bettencourt 2016 iGEM team improvement

Introduction

In the Frank&Stain project of the Paris Bettencourt 2016 iGEM team we produced synthetic enzymes to remove red wine stains from fabric. These enzymes are designed to replace perchloroethylene (PERC), a toxic solvent used in dry cleaning that will soon be banned in France. Moreover, we have identified 40 short peptides with affinity for cotton, linen, wool, polyester and silk using the method of phage display. These Fabric Binding Domains (FBDs) were fused to our enzymes with the aim of improving their efficiency and specificity for removing wine stains.
Before attaching our FBDs to the enzymes we had to validate their specificity and binding strength, so we:

  • fused FBDs to GFP
  • overexpressed the fusion proteins in E. coli
  • obtained the cell extracts with our overexpressed GFP-FBDs
  • tested the specificity and strength of binding of FBDs to different fabrics, by measuring levels of fluorescence after washing the fabrics

  • To make our results more meaningful and relevant we have used this BioBrick, sfGFP fused to CBDcex created by 2014 Imperial College London team, as a positive control.


    Results

    We improve the characterization of this part by testing it in different fabrics. In addition to cellulose we also tested the binding strength of this part to cotton, linen, silk and wool, because in the context of our project these fabrics were most convenient.
    This part was used in our experiments as a positive control for both expression and binding, since we already knew that the part had worked in previous experiments done by Imperial College London 2014 iGEM team as by Edinburgh 2015 iGEM team.
    In our case, as we can see in figures 1 and 2, the experience with this part was very positive. It was expressed successfully, with preserved function and high affinity for cellulosic fabrics, such as cotton and linen.

    CBD.jpg
    Figure 1 Analysis of BBa_K1321357 Biobrick registry part. Cell extract from E. coli expressing this part was incubated overnight in a 96 well plate with fabric placed at the bottom of the wells. Water, PBS, 5% BSA, 70% Ethanol and Catechol 30mM were the washing solutions used in the experiment to remove the non-bound CBD, as well as to test its binding strength. The data displayed corresponds to the values after the final wash, normalised using the values from the first wash. The intensity of the colour corresponds to the GFP signal measured at that point. Excitation 475nm and emission 515nm.


    PRESENTATION.jpg
    Figure 2 Bar plot showing the fluorescence of cell extracts relative to the control (No GFP expressed). Here we compare the fluorescence of different cell extract expressing Fabric Binding Domains (FBDs) with affinity for different fabrics. The part BBa_K1321357 (GFP-CBDex) was used as positive control. The blue bar correspond with the negative control (no GFP in the cell extract) whereas the green ones with those whose fluorescence was higher than the control and in red those which presented no fluorescence, maybe because lacking activity or functionality.


    References:

    Buchert, J., Pere, J., Johansson, L. S., & Campbell, J. M. (2001). Analysis of the surface chemistry of linen and cotton fabrics. Textile Research Journal, 71(7), 626-629.

    Goldstein, M. A., Takagi, M. A. S. A. H. I. R. O., Hashida, S. E. I. I. C. H. I., Shoseyov, O. D. E. D., Doi, R. H., & Segel, I. H. (1993). Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A. Journal of bacteriology, 175(18), 5762-5768.

    Francisco, J. A., Stathopoulos, C., Warren, R. A., Kilburn, D. G., & Georgiou, G. (1993). Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains. Bio/technology (Nature Publishing Company), 11(4), 491-495.



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