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

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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. <br> <br>
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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. <br> <br>
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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). <br> <br>
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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. <br> <br>
  
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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. <br> <br>
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In order to perform the skim milk plate test, colonies of KerUS and colonies of DH5⍺ 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 DH5⍺ 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 DH5⍺ samples. The success with this test proved that the cells containing the plasmids were successfully secreting the KerUS enzyme! <br> <br>
  
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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). <br> <br>
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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. <br> <br>
  
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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. <br> <br> <br>  
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The hair degradation assay aimed to repeat the success of the skim milk plates in a quantitative manner.  KerUS plasmid-containing cultures, and DH5⍺ 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.<br> <br>
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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. <br> <br>
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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.  <br> <br>
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Both 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.<br> <br>
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<li>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 </li>
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</ol> <br> <br> <br>  
 
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Revision as of 06:00, 19 October 2016

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 DH5⍺ 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 DH5⍺ 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 DH5⍺ 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.

The hair degradation assay aimed to repeat the success of the skim milk plates in a quantitative manner. KerUS plasmid-containing cultures, and DH5⍺ 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.

Both 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.

  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