Part:BBa_K1717000:Design

Keratinases are isolated from several bacterial and fungal species, but have been expressed in E. coli with more limited success. The bacterial chassis that we are using is a strand of E-coli called K-12. Like all E. coli, this strain is gram-negative. After extensive research and looking at the projects of Chicago, Sheffield and Taiga, we deduced that the signal peptides (which were those of a gram-positive source organism) could be causing the poor secretion of the enzyme in those projects. Previous teams either left the gram-positive signal peptides intact, or removed signal peptide sequences entirely. Our approach was instead to remove the original signal peptide sequence and replace it with one optimized for secretion in E. coli (pelB, with the sequence taken from part Part BBa_J32015) The part is designed to express the keratinase when the promoter is induced, and secrete it into the periplasm, where some will escape into the media extracellularily. DNA sequences were compared used Clustal to ensure protein coding sequences were not altered, and that start and stop codons were correct. NEBCutter V2.0 was utilized to check for illegal cut sites. One EcoRI site and one PstI site were identified, and a silent mutation was introduced in order to remove them.