Difference between revisions of "Team:OLS Canmore/Proof"

PROOF OF CONCEPT

With the complete version of our biobrick, the next step for our team 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 project, our team ran two separate assays this year as a proof of concept. The first of these two was a skim milk plate assay, a qualitative test that, if successful, would visibly show the presence of any proteolytic enzymes secreted by our cells, as keratinase is capable of breaking down casein protein found in milk. 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, two overnight colonies containing KerUS, as well as two DH5⍺ colonies were grown in LB broth in preparation for lysing. After all tubes received ampicillin, half of the tubes (one of the DH5⍺, and one containing KerUS) were induced with IPTG, while the other half simply received distilled water. After the lysing of the cells, the supernatant was collected from all tubes and was saved. Skim milk plates were then prepared by mixing agar and skim milk powder, as well the addition of ampicillin. The four samples of supernatant, two that had been induced with IPTG and two that had not, were pipetted onto our skim milk plates. After an observation period of 5 days, our team observed multiple zones of clearing around both of our KerUS samples. Our success with this test proved that the cells containing our biobrick was successfully secreting the KerUS enzyme.

Our hair degradation assay, similarly, aimed to repeat the success of the skim milk plates in a quantitative way. First, two cultures of each E.coli JM109 with the kerA plasmid, with the kerUS plasmid, and without any plasmid, were grown overnight. Dried hair samples, weighing 0.05 grams each, were then placed into the tubes of culture. After a incubation period of 5 days, the hair was then removed, dried, and weighed to record the final mass. Unfortunately, this test was not as successful, as no significant change in 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 slight discoloration in both the culture and the plastic tubes. The discoloration was a faint pink colour; very similar to the lids of our tubes which could be evidence of some proteolytic activity (can you please explain this a little bit more).

Both our results suggest the presence of some possible enzyme activity. Although the hair degradation test was not as successful, the discoloration could very well be because our enzyme is breaking down proteins found in the dye of the cap. Regardless, the results of both of these tests have huge implications for our project. These two tests marked the first time our team tested our project for proteolytic properties since starting over 3 years ago. If these results are to be believed, our project is capable of breaking down hair, and perhaps could one day be successfully implemented as a solution to keratin waste in wastewater treatment and the poultry industry.

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