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

Our team will create two different keratinase-expressing E. coli systems. One will express Keratinase A (KERA), an enzyme optimized to degrade hair, and the second will express Keratinase US (KERUS).

The KERA and KERUS sequences will be optimized for expression in E. coli to prevent the misfolding of the protein in the periplasm, and allow for the export tag to be cleaved so the protein can leave the cell. The composite part will be synthesized into standard biobrick form with an RFP vector (part BBa_J04450), for submission to the iGem parts registry. An IPTG-inducible promoter (part BBa_J04500) from the standard registry of parts will then be added to each KER gene create an expression cassette for this protein, also for submission to the iGem parts registry. We will then work to characterize our genes and optimize protein expression and function.

Plan Part 1 - Creating Basic Coding Sequence Biobricks:

1. KERA and KERUS coding sequences synthesized into plasmids, thanks to our sponsors at BioBasic.
2. Obtain parts BBa_J04450 (RFP Cassette), from IGEM registry.
3. Restriction digest: RPF was cut at the sites EcoRI and SpeI. KERA and KERUS plasmids cut with XbaI and PstI
4. Ligation: Ligated our KERA and KERUS inserts into our BBa_J04450 (RFP) vector plasmid rings. A quick test for success - re-ligated RFP plasmids would glow red in transformation, while newly ligated plasmids will not glow red.
5. Transform new constructs into competent E. coli and grow on CHLOR plates to test for correct ligation.

Plan Part 2: Creatin KerA/US Expression Cassette Biobricks

1. KERA and KERUS coding sequences synthesized into plasmids, thanks to our sponsors at BioBasic.
2. Obtain parts BBa_J04500 (IPTG-Inducible promoter), from IGEM registry.
3. Restriction digest: Promoter Part was cut at the sites SpeI and PstI. KERA and KERUS plasmids cut with XbaI and PstI
4. Ligation: Ligated our KERA and KERUS fragment inserts into our promoter vectors.
5. Transform new constructs into competent E. coli and grow on CHLOR plates to test for correct ligation.