Team:OLS Canmore/Proof

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PROOF OF CONCEPT

With the complete version of our biobrick, the next step was to test the construct. In order to demonstrate the presence of any proteolytic activity, as well as to test the practicality and efficiency of our constructs, we ran two separate assays to provide a proof of concept. For both of the following tests, we used a “non-biobrick” version of our plasmid as we had not yet inserted that part into the standard backbone (it was in pUC57 instead of pSB1C3) for submission to iGEM.

The first of these two was a skim milk plate assay: a qualitative test that, if successful, would visibly show evidence of keratinase activity in the form of zones of clearing. These zones of clearing are created by the enzyme of degrading the casein found in milk (1).

Second was the hair degradation assay, a semi-quantitative test that was more relevant to our goals. The test is done by putting a small amount of hair into a tube containing cultured cells containing our biobrick.

In order to perform the skim milk plate test, colonies of KerUS and colonies of JM109 with no Ker plasmid were grown-up and lysed. The supernatant from the lysis was plated on skim milk plates. Non-lysed culture of KerUS plasmid-containing bacteria and JM109 with no Ker plasmid were also plated. After an observation period of 5 days, our team observed multiple zones of clearing around KerUS samples, and not around the negative-control JM109 samples. The success with this test proved that the cells containing the plasmids were successfully secreting the KerUS enzyme!

For more information on creating skim-milk plates, lysing cells, and plating on skim-milk agar can be found on the ‘Experiments’ page. A more in-depth analysis of the results can also be found on the ‘Results’ page.

Figure 1. Shows a zone of clearing around the second day of the assay. The zone of clearing is on a quadrant that was plated with KerUS lysis supernatant.

Figure 2. Shows a zone of clearing around the plated non-lysed KerUS plasmid-containing cultures.

Figure 3. Shows a zone of clearing around lysed colonies that contained the KerUS plasmid.

The hair degradation assay aimed to repeat the success of the skim milk plates in a quantitative manner. KerUS plasmid-containing cultures, and JM109 cultures used as a negative control were induced with IPTG. Then 0.05g of hair was placed into each culture, and these were incubated for 5 days. More information on this assay can be found on the ‘Experiments’ page of the wiki.

Unfortunately, this test was not as successful, as no significant change in hair mass was found. We suspected that this could possibly due to the fact that we could not keep the cells at optimal pH and temperature.

However, our team did observe discoloration in both the culture and the plastic tubes. The discoloration was of a faint pink colour; very similar to the lids of our tubes. We hypothesize that this is because the enzyme in our tubes was actually breaking down proteins found in the lids, most likely in the dye, thus causing the discoloration. Unfortunately, we have been unable to find any reliable information of the materials used in the production of the tubes.

Figure 4. Shows the pink discoloration of the falcon tubes used in the experiment.

After discovering an error in the antibiotic resistance with the synthesis of our parts (for more explanation please see the 'Results' page), we were able to set up some last-minute proof of concept assays. These specific assay were performed by our mentor Lisa as she has access to a lab more often than we do. However, a the time of writing this page for the wiki, we have already begun our own replicate assays, but do not have results to share yet. We do however plan to show these during the presentation and poster sessions in Boston.
Lisa was able to start some liquid-culture feather degradation assays. To simplify, she cultured keratinase expression cassette-containing plasmids in LB-broth, induced with IPTG, and added feathers to them. As a control, cells without the keratinase expression cassette, and cells that were not induced with IPTG were used. At least one culture from each group was incubated at 37 degrees Celsius, and one at 30 degrees celsius. After 18 hours of incubation, the tubes containing the induced IPTG promoter+keratinase cultures became significantly cloudier which is demonstrative of keratin-degradation. Pictures and more detailed results for this assay can be found on the 'Results' page.

These results suggest the presence of some enzyme activity. Although the hair degradation test was not as successful, the discoloration may be a result of keratinase breaking down proteins found in the dye of the cap. Regardless, the results of both of these tests have huge implications for our project moving forward. These two tests marked the first time our team was able to test for enzyme activity in the 2 years of the project. With future characterization and optimization of the environment, the project may be capable of breaking down hair and feathers for processing into useful products.

Citations:

1. Gupta R, Tiwary E, Sharma R, Rajput R, Nair N. Microbial Keratinases: Diversity and Applications. Thermophilic Microbes in Environmental and Industrial Biotechnology [Internet]. New York; Springer; n.d. [cited 2016 October 12]. 951p. Available from: http://www.springer.com/us/book/9789400758988

Contact us at:
https://www.facebook.com/OLeSsence/
@igem_canmore
larvisais@redeemer.ab.ca