Difference between revisions of "Part:BBa K2020003"

Revision as of 20:33, 8 October 2016

mutated expression system for subtilisin E in E. coli (S221Y)

Once introduced into E. coli, this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. By performing a site-directed mutagenesis, serine in the catalytic triad of the enzyme was exchanged against tyrosine. As a consequence, the enzyme looses its proteolytic activity.

Usage and Biology

Subtilisin E is an alkaline serine protease which non-specifically digests proteins. It is naturally produced by Bacillus subtilis.

This composite part consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for Escherichia coli codon usage (BBa_K2020000).

This mutated version of the BioBrick BBa_K2020002 was created to prove that serine²²¹ in the catalytic triad of subtilisin E is essential for the activity of the enzyme.

Sequence and Features