Difference between revisions of "Part:BBa K3153002"
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<h1>Background </h1>
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<h2>Background </h2>
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Metallothionein (MT) plays an important role in bioremediation of heavy metals. MT is a kind of small molecular peptide rich in cysteine (Cys-) residues with low molecular weight. Its Cys- residues can adsorb many heavy metals such as lead, mercury and cadmium. However, since arsenic is a metalloid, these MTs have no specific adsorption on it.
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Recently, scientists isolated and identified an MT from a species of arsenic-tolerant Marine algae (Fusus vesiculosus) that, in vitro, demonstrated a high affinity for arsenic binding capacity.
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In this project, we used the bacterial surface display technology to display fMT on the bacterial surface, and thus enhance the ability of absorbing arsenic.
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Inspired by HUST-China team in 2014, the fusion protein oprf-fMT was constructed and heterogeneiously expressed by the E. coli expression system pET series plasmid.
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<h2>Protocol </h2>
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<h2>Result</h2>
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As shown in figure, the photograph of SDS-PAGE and Western blot of orpF-fMT fusion protein was successfully expressed under IPTG induction with the molecular weight between 25KDa and 35kda, which was consistent with the expected molecular weight of 27.4kda, indicating that the fusion protein was successfully expressed.
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https://static.igem.org/mediawiki/parts/0/00/T--Worldshaper-Wuhan--parts.png
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Fig.1 IPTG-induced protein expression of recombinant plasmid pET24a-oprF-fMT in SDS-PAGE M：protein marker, 1-3 for oprF-fMT fusion protein expression, and 1: IPTG(-); 2: IPTG (1mM), 37℃,6h; 3: IPTG(1mM), 25℃,overnight; (b) IPTG-induced protein expression of recombinant plasmid pET24a-oprF-fM in Western blot by using anti-Histag antibody.

Revision as of 07:24, 20 October 2019


oprF-fMT

A surface-displayed fusion protein for aresenic removal. OprF, the major outer membrane (OM) protein of Pseudomonas aeruginosa, has been used for presentation of foreign protein on the cell surface. fMT,is an arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga Fucus vesiculosus

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 608
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 697
  • 1000
    COMPATIBLE WITH RFC[1000]


Background

Metallothionein (MT) plays an important role in bioremediation of heavy metals. MT is a kind of small molecular peptide rich in cysteine (Cys-) residues with low molecular weight. Its Cys- residues can adsorb many heavy metals such as lead, mercury and cadmium. However, since arsenic is a metalloid, these MTs have no specific adsorption on it.

Recently, scientists isolated and identified an MT from a species of arsenic-tolerant Marine algae (Fusus vesiculosus) that, in vitro, demonstrated a high affinity for arsenic binding capacity.

In this project, we used the bacterial surface display technology to display fMT on the bacterial surface, and thus enhance the ability of absorbing arsenic.

Inspired by HUST-China team in 2014, the fusion protein oprf-fMT was constructed and heterogeneiously expressed by the E. coli expression system pET series plasmid.


Protocol


Result

As shown in figure, the photograph of SDS-PAGE and Western blot of orpF-fMT fusion protein was successfully expressed under IPTG induction with the molecular weight between 25KDa and 35kda, which was consistent with the expected molecular weight of 27.4kda, indicating that the fusion protein was successfully expressed.

T--Worldshaper-Wuhan--parts.png

Fig.1 IPTG-induced protein expression of recombinant plasmid pET24a-oprF-fMT in SDS-PAGE M：protein marker, 1-3 for oprF-fMT fusion protein expression, and 1: IPTG(-); 2: IPTG (1mM), 37℃,6h; 3: IPTG(1mM), 25℃,overnight; (b) IPTG-induced protein expression of recombinant plasmid pET24a-oprF-fM in Western blot by using anti-Histag antibody.