Part:BBa_K1520507

OMPA-golB

OMPA-golB A protein to catch gold ions. OMPA present golB outside the membrane and golB will catch gold ions.

GolB is a identified small gold-specific protein in S. typhimurium that is highly induced in the presence of Au(I) over Cu(I), Ag(I), Zn(II), or Hg(II) ions. It has less than 70 amino acids in length and contains a similar metal binding motif, MTCXXC, to that of the previously reported human copper chaperone AtoX1 and yeast copper chaperone AtX1.

We display GolB on E. coli cell surface by fusing it with OmpA, an E. coli outer membrane protein to increase the disposing efficiency. The Lpp–OmpA fusion system has been previously used to display proteins as large as 263 amino acids on the surface of E. coli. Following a similar protocol, we constructed a plasmid carrying the OmpA–GolB expression gene by connecting the N-terminal 1–159 amino acids of OmpA with the full-length GolB (Fig. 1a)

a) An illustration of the gold(I)-specific protein GolB displayed on E. coli cell surface via the outer membrane protein OmpA. b) SDS-PAGE and immunoblotting analyses of surface-displayed GolB protein. The black arrow on the SDS-PAGE gel indicates the OmpA–GolB protein expressed in bacterial membrane fraction from induced bacteria expressing OmpA–GolB (lane 4). The membrane fraction from uninduced bacteria (lane 3), the supernatant protein fraction from induced (lane 2) or uninduced (lane 1) bacteria are used as controls. The presence of OmpA–GolB protein was further confirmed by immunoblotting analysis using anti-HA and anti-FLAG antibodies. c) Immunofluorescence labeling of E. coli cells using anti-FLAG antibody and FITC conjugated anti-mouse IgG antibody.

Metal selective adsorption of GolB displayed E. coli. E. coli (DH10B) strains containing OmpA-GolB plasmid (+) or not (-) were induced by 0.002% arabinose (black columns) or not (white columns), then grown overnight in LB with an addition of 50 μM HAuCl4 (Au3+) or 50μM KAu(CN)2 (Au+) or 50μM AgNO3 (Ag+) or 50μM CuSO4 (Cu2+).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 770
  Illegal XhoI site found at 742
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]