Part:BBa_K2020002

expression system for subtilisin E in E. coli

Once introduced into Escherichia coli, this BioBrick is able to produce subtilisin E and simultaneously secret the enzyme into the periplasm of the cell.

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).

For the varification of the function of this part, a skim milk assay on agar plates was performed. Therefore, LB skim milk agar plates containing IPTG and the needed antibiotic were poured and the E. coli BL21 cells containing the plasmid with the expression system were streaked out.

The figure shows the results of a skim milk assay. This assay utilizes the clearance of skim milk due to proteolysis to detect proteolytic activity. Here, cells containing an empty backbone (right) and cells containing the expression system for native subtilisin E (left) after incubation for 3 days at 30°C can be seen.

The cells carrying only an empty backbone don't seem to cause the medium to become transparent. A clearance of the skim milk can only be detected for cells containing the expression system for native subtilisin E. Thus, a proteolytic activity can be deduced. In conclusion, the biobrick BBa_K22020002 causes the expression of native subtilisin E in E. coli cells.

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]