Difference between revisions of "Team:OLS Canmore AB CA/Results"

This year our team started what is projected to be a multi-year and multi-step project. Successfully completed protocols this year include:

1. Rehydration of registry DNA
2. Transformation of registry DNA and synthesized plasmids (KerA and KerUS) into competent cells.
   • Evidence of this working includes successful growth of these colonies on AMP plates, after transforming a plasmid with AMP resistance into these cells. Negative controls grown did not grow.
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3. Growing cultures from single colonies
   • The broth was not cloudy at first, but once the protocol was completed, the broth was cloudy, therefore the protocol worked. Once plated, colonies grew.
4. Miniprep
   • Our spectrophotometer confirmed the presence of DNA in our miniprep columns, and when digested, DNA bands were visible via gel.
5. Restriction digest
   • Our gel confirms that DNA is present in our restriction digest samples, however resolving bands of DNA proved challenging. The size bands that were faintly visible did not appear to be the appropriate size to match DNA bands expected.
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In the Future:

Immediate steps will involve troubleshooting our restriction digest protocol to successfully get our constructs into the standardized biobrick format, for submission to the parts registry. Ligation will be required, which we have not yet attempted, followed by transformation, which we have had good success with in our lab to date.

In the longer term, we plan to focus on the implementation and industrialization of our project. There are many things that we must look at in order to successfully optimize and implement our construct. Our steps to ensure that our construct works will be to run an initial qualitative assay to determine if the keratinase is in fact breaking down hair and feathers. If initial qualitative results are positive, a quantitative assay will determine how much hair and feathers can be broken down and at what speed.

Our third step will be to optimize our construct for usage in mass quantities in the real world and for utilization in a bioreactor. We will test our construct so that we may find the optimal enzymatic concentration, co-factors, inhibitors, metal ion cross-reactions, and anything else that may impact degradation of hair and feathers.

We are also going to research how current bioreactors in wastewater treatment facilities could be modified to work with our construct, and ensure safety in containment. This will go hand in hand with our lab work as optimization test will teach us how keratinase can also be inhibited and denatured, allowing as a type of kill switch. This lab work and research will also be applied to the implementation of our project in order to manage feather waste.