Part:BBa_K2020006

mutated expression system for subtilisin E in E. coli (Y77X)

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). Once introduced into E. coli, this BioBrick is able to produce a modified version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins. By performing a site-directed mutagenesis, tyrosine⁷⁷ in the propeptide of the enzyme was exchanged against the amber stop codon UAG. Therefore, the expression of the enzyme is interrupted.

Usage and Biology

This composite part was created to integrate a non-canonical amino acid into the sequence of the propeptide 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 280
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

biology	Bacillus subtilis
chassis	Escherichia coli
protein	subtilisin E