Part:BBa_K2372003

beta-lactam synthetase double mutant

The BLS mutant is a rationally designed mutant of the β-lactam synthetase to increase substrate range and codon-modified to ensure correct DNA synthesis. β-lactam synthetase (BLS), found in Streptomyces clavuligerus, catalyzes the formation of the β-lactam ring of clavulanic acid in an ATP and Mg²⁺ dependent manner by intramolecular closure. In our project we were using BLS for activating our chemical synthesis by formation of a β-lactam ring in vitro. In order to simplify purification a thrombin cleavage side and 6xhis-tag was added at the end of the enzyme sequence.

BLS mutant
Function	Wide range β-lactam synthetase
Use in	Prokaryotic cells
RFC standard	RFC 10
Backbone	pSB1C3
Organism	Streptomyces clavuligerus
Source	IDT (DNA synthesis)
Submitted by	Tuebingen 2017

References
Bachmann, B. O., Li, R., & Townsend, C. A. (1998). beta-Lactam synthetase: a new biosynthetic enzyme. Proc Natl Acad Sci U S A, 95(16), 9082-9086.

Miller, M. T., Bachmann, B. O., Townsend, C. A., & Rosenzweig, A. C. (2001). Structure of beta-lactam synthetase reveals how to synthesize antibiotics instead of asparagine. Nat Struct Biol, 8(8), 684-689. doi:10.1038/90394

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 AgeI site found at 9
  Illegal AgeI site found at 199
  Illegal AgeI site found at 379
  Illegal AgeI site found at 544
  Illegal AgeI site found at 1036
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 631
  Illegal BsaI site found at 1224