Team:OLS Canmore/Basic Part

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BASIC PARTS

Keratinases in nature are a type of proteolytic enzymes which means they break down proteins into smaller units. While keratinases have been isolated within several different fungal and bacterial species, they have not been commonly expressed in E. coli which are gram negative. For our project, we are using a strand of E. coli called DH5alpha as our bacterial chassis which allow us to express protein at a relatively high rate and to work more easily work in the lab.

By looking at the projects of Chicago, Sheffield, we came to the conclusion that the signal peptides, from the Bacillus isolated sequence gram positive, were the source to poor secretion of the enzyme. We believe this was because Chicago and Sheffield did not take in account the different number of membranes of E. coli as compared to Bacillus therefore they did not optimize it to be gram negative. In our project, we decided to remove the original signal peptide and replace it with a peptide that is optimized for the secretion of E. coli. Once the part is paired with an IPTG promoter, the part is designed to express Keratinase and it is able to secrete keratinase into the space between the membranes. However, some of the Keratinase will naturally escape into the media. We made sure that the start and stop codons were correct, and that the protein coding sequences were not altered by comparing the DNA sequences using Clustal. We also used NEBCutter V2.0 to check for any illegal cut sites and we discovered that there was one EcoR and one Pstl site identified. In order to remove these illegal cut sites, a silent mutation was introduced to them.

Keratinase A (BBa_K1717171)
Keratinase A is found most active in degrading the protein keratin in hair. It is a basic parts part that is compromised from a synthesized Keratinase A coding sequence which is optimized for expression in E. Coli.

Keratinase US (BBa_K1717172)
Keratinase US is a type of keratinase that comes from the Bacillus bacteria. Keratinase US is a basic part parts comprised from a synthesized KERUS protein coding sequence, that is optimized for expression in E.coli. Keratinase US is most active in degrading keratin in feathers.