Difference between revisions of "Part:BBa K3153002"

Latest revision as of 02:27, 22 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.

Fig.1 Schematic diagram of oprF-fMT fusion protein expressed in pET system

Fig.2 Genetic circuit of oprF-fMT fusion protein expression

Protocol

IPTG induction and protein sample preparation

Constructed plasmid pET24a(+)-orpF-linker-fMT and pET24a(+)-SpPCS was transformed into E.coli BL21(DE3) strain. Single colony was selected to inoculate LB broth containing kanamycin and cultured overnight. Then overnight culture was inoculated in fresh LB broth containing kanamycin at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.6-0.8. IPTG was added to the bacterial liquid for induction, so that the final concentration of IPTG was 1mM,cultured at 25℃ overnight.After induction,the bacteria were centrifuged, and the bacteria pellet was treated with 5* SDS-PAGE loading buffer and boiled for 5-10min for the preparation of protein samples.

SDS-PAGE

A 12% SDS-PAGE gel was prepared, as the expected molecular weight of oprF-fMT fusion protein was about 25Kda. Then 5μl marker or 20μl protein samples were loaded in each lane. Run SDS-PAGE and the electrophoresis was complete when the the dye front migrates about 2mm from the bottom of the gel. The gel was stained with Coomassie brilliant blue for 30min, then then destained in destain buffer overnight.

WB

A 12% SDS-PAGE gel was prepared and 5μl marker or 20μl protein samples were loaded in each lane. At the end of SDS-PAGE, the gel was transferred to PVDF membrane in WB transfer buffer for 2h. Then the PVDF membrane was washed in TBST and blocked in WB blocking buffer for 1h. After blocking, the membrane was washed in TBST and incubated with Rabbit-anti-Histag antibody diluted 1:10,000 in blocking buffer at 4℃overnight. The next day the membrane was washed in TBST three times and incubated with goat-anti-rabbit IgG conjugated with HRP diluted 1:1000 in blocking buffer at RT for 1h. After washing three times in TBST, the membrane was exposed in the darkroom with film developer solution and the WB photos was obtained.

Results

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.

Fig.3 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.

@@ Line 27: / Line 27: @@
 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.
 https://static.igem.org/mediawiki/parts/f/fa/T--Worldshaper-Wuhan--parts6.jpeg
+Fig.1 Schematic diagram of oprF-fMT fusion protein expressed in pET system
 https://static.igem.org/mediawiki/parts/e/ed/T--Worldshaper-Wuhan--parts5.jpeg
+Fig.2 Genetic circuit of oprF-fMT fusion protein expression
 <h2>Protocol </h2>
@@ Line 37: / Line 42: @@
 IPTG induction and protein sample preparation
-Constructed plasmid pET24a(+)-orpF-linker-fMT 和pET24a(+)-SpPCS was transformed into E.coli BL21(DE3) strain. Single colony was selected to inoculate LB broth containing kanamycin and cultured overnight. Then overnight culture was inoculated in fresh LB broth containing kanamycin at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.6-0.8. IPTG was added to the bacterial liquid for induction, so that the final concentration of IPTG was 1mM,cultured at low temperature induction, 25℃, and overnight culture.After induction， the bacteria were centrifuged, and the bacteria pellet was treated with 5* SDS-PAGE loading buffer and boiled for 5-10min for the preparation of protein samples.
+Constructed plasmid pET24a(+)-orpF-linker-fMT and pET24a(+)-SpPCS was transformed into E.coli BL21(DE3) strain. Single colony was selected to inoculate LB broth containing kanamycin and cultured overnight. Then overnight culture was inoculated in fresh LB broth containing kanamycin at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.6-0.8. IPTG was added to the bacterial liquid for induction, so that the final concentration of IPTG was 1mM,cultured at 25℃ overnight.After induction,the bacteria were centrifuged, and the bacteria pellet was treated with 5* SDS-PAGE loading buffer and boiled for 5-10min for the preparation of protein samples.
 SDS-PAGE
-A 12% SDS-PAGE gel was prepared, as the expected molecular weight of oprF-fMT fusion protein was 20Kda. Then 5μl marker or 20μl protein samples were loaded in each lane. Run SDS-PAGE and the electrophoresis was complete when the the dye front migrates about 2mm from the bottom of the gel. The gel was stained with Coomassie brilliant blue for 30min, then then destained in destain buffer overnight.
+A 12% SDS-PAGE gel was prepared, as the expected molecular weight of oprF-fMT fusion protein was about 25Kda. Then 5μl marker or 20μl protein samples were loaded in each lane. Run SDS-PAGE and the electrophoresis was complete when the the dye front migrates about 2mm from the bottom of the gel. The gel was stained with Coomassie brilliant blue for 30min, then then destained in destain buffer overnight.
 WB
@@ Line 54: / Line 59: @@
 https://static.igem.org/mediawiki/parts/0/00/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.
+Fig.3 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.