Difference between revisions of "Part:BBa K1520507"

Latest revision as of 00:33, 17 October 2014

OMPA-golB

OMPA-golB It is 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.

a) An illustration of the gold-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.

1. uninduced bacteria are used as controls.

2. the supernatant protein fraction from induced bacteria.

3. bacteria membrane fraction from uninduced bacteria.

4. bacteria membrane fraction from induced bacteria expressing OmpA–GolB (The black arrow on the SDS-PAGE gel indicates the OmpA–GolB protein expressed)

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.(+)stands for containing OmpA-GolB plasmid, and (-) stands for not containing the plasmid. black stands for bacteria induced by 0.002% arabinose and the white ones were not induced by arabinose.

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]

@@ Line 3: / Line 3: @@
 OMPA-golB
-A protein to catch gold ions. OMPA present golB outside the membrane and golB will catch gold ions.
+It is 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)
+We display GolB on E. coli cell surface by fusing it with OmpA, an E. coli outer membrane protein to increase the disposing efficiency.
 [[File:golB.jpg]]
+a) An illustration of the gold-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.
+. uninduced bacteria are used as controls.
+. the supernatant protein fraction from induced bacteria.
+. bacteria membrane fraction from uninduced bacteria.
+. bacteria membrane fraction from induced bacteria expressing OmpA–GolB (The black arrow on the SDS-PAGE gel indicates the OmpA–GolB protein expressed)
+c) Immunofluorescence labeling of E. coli cells using anti-FLAG antibody and FITC conjugated anti-mouse IgG antibody.
+[[File:golB2.jpg]]
+Metal selective adsorption of GolB displayed E. coli.(+)stands for containing OmpA-GolB plasmid, and (-) stands for not containing the plasmid. black stands for bacteria induced by 0.002% arabinose and the white ones were not induced by arabinose.
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