Part:BBa_K2020003

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

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 an inactive 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, serine in the catalytic triade of the enzyme was exchanged against tyrosine. Therefore, the enzyme looses its proteolytic activity.
With this composite part iGEM Aachen 2016 was able to prove that serine²²¹ in the catalytic triade of subtilisin E is essential for the activity of the enzyme.

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]