Part:BBa_K1717173:Design
Keratinase US under control of LacI promoter.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 279
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 297
Design Notes
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, and no such sites were identified.
Source
The sequence for the KERUS coding region was taken from the Uniprot gene/protein sequence bank. KerUS: http://www.uniprot.org/uniprot/A0A059PWB6