Part:BBa_I757011

bla_frag1(aa 26-199) Fusion Part

• first fragment of the split enzyme TEM 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 second half of split lactamase (part BBa_I757012)
• this fragment contains stabilizing mutations
• NgoMIV / AgeI protein fusion part
• iGEM Team Freiburg 2007
• Synthetic DNA by GeneArt
• Part in pGA4 vector (AmpR, ColEI ori)
• Part contains amino acids 26-199 of TEM-116 lactamase according to the numbering of Ambler, or amino acids 24-197 according to consecutive numbering with signal sequence.

Mutations: V31A, R120G, H153R, M182T (positions according to Ambler consensus)

V31A, located in the N-terminal half of helix H1, identified in a mutant 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), increases intrinsic helix propensity.

R120G, located at the N-terminus of helix H4, identified in mutants after genetic selection in the terminal truncation and circular permutation backgrounds, alleviates repulsive forces with helix macrodipole.

H153R, located at the C-terminus of helix H6, identified in mutants after genetic selection in the terminal truncation and circular permutation backgrounds, converts residue 153 to consensus.

M182T, located at the N-cap position of helix H8, identified as global suppressor mutation (Huang et al., 1997; Siederaki et al., 2001), dominant exchange in genetic selection for terminal truncation-suppressor mutations, converts residue 182 to consensus, mechanism unclear.

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 535
  
• 1000
  COMPATIBLE WITH RFC[1000]