Part:BBa_K2020004:Design

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

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]

Design Notes

The sequence of the subtilisin E gene from Bacillus subtilis was codon optimized for E. coli with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon AGC of serine²²¹ was substituted with the stop codon TAG.

Source

The sequences of the BioBrick parts were obtained through the Registry.

part	BioBrick No.
LacI promoter	BBa_R0010
RBS	BBa_B0034
CDS	BBa_K2020001
	BBa_J32015	BBa_K2020000
terminator	BBa_B0010

The construct was ordered at IDT.