Part:BBa_I757012

bla_frag2 (aa 200-290) Fusion Part

• second fragment of the split enzyme beta-lactamase
• purpose: split lactamase activity can be recovered when halves are brought in close proximity by e.g. fusion to interacting domains
• works with first half of split lactamase (part BBa_I757011)
• this fragment contains stabilizing mutations
• NgoMIV / AgeI protein fusion part
• iGEM Team Freiburg 2007 and Jochen Hecky
• Synthetic DNA by GeneArt
• Part in pGA4 vector (AmpR, ColEI ori
• part contains amino acids 200-290 of TEM-116 lactamase with numbering according to Ambler, or amino acids 198-286 according to consecutive numbering with signal sequence

Split enzyme: the split site has been shifted by two aa compared to previous publications by the groups of S. Michnik and H. Blau.

Mutations: L201P, I208M, E212K, A224V, R275L (positions according to Ambler consensus)

L201P, located at N-terminus of helix H9, identified in a mutant after genetic selection for mutations compensating interface-disrupting mutations (Jochen Hecky, unpublished results).

I208M, located in the C-terminal half of helix H9, identified in mutants after genetic selection for suppressor mutations in the terminal truncation and circular permutation backgrounds, presumably improves van der Waals contacts across the domain interface.

E212K, located at C-terminus of helix H9, identified in a mutant after genetic selection for mutations compensating defects induced by circular permutation, presumably creates favourable electrostatic interaction to D209.

A224V, resides on second crossover loop, identified in mutants after genetic selection for exchanges compensating defects induced by removal of the first five amino acid residues of the mature TEM-1 protein (Hecky & Müller, 2005), improves hydrophobic contacts between crossover loop, C-terminal helix H11 and the underlying beta-sheet, reinforces domain interface.

R275L, located in N-terminal half of C-terminal helix H11, identified in a mutant after genetic selection for mutations compensating defects induced by circular permutation (Osuna et al., 2002), presumably acts by improving hydrophobic contacts between the C-terminal helix H11 and the beta-sheet underneath.

Part contains one additional Gly at the N-terminus after AlaGly coded by the NgoMIV restriction site as short linker for fusion constructs.

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 4
  Illegal AgeI site found at 280
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 137