Difference between revisions of "Part:BBa K2020004"

Latest revision as of 23:47, 19 October 2016

mutated expression system for subtilisin E in E. coli (S221X)

Once introduced into Escherichia coli, this BioBrick is able to produce a modified 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 the amber stop codon UAG. As a consequence, the expression of the enzyme is interrupted.

As the referring proof of principle of this part, documented for the BioBrick K2020003, of exchanging serine against a larger amino acid namely tyrosine worked, it can be assumed, that this part will also operate correctly.

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 E. coli codon usage (BBa_K2020000).

This mutated version of the BioBrick BBa_K2020002 was created to integrate a non-canonical amino acid into the sequence of subtilisin E by adding an orthogonal tRNA/aminoacyl-synthetase pair that is capable of incorporating the non-canonical amino acid of choice in response to the UAG stop codon.

Sequence and Features