Difference between revisions of "Part:BBa K3153001"

Revision as of 13:55, 21 October 2019

SpPCS

Phytochelatin synthase from a yeast, Schizosaccharomyces pombe

Sequence and Features

Background

An important protein called phytochelates (PC) with a high affinity for arsenic has been found. PC is a natural peptide in plants and fungi, the general formula of the structure is (γ-Glu-Cys-)n-Gly(n=1-11) . It was found that PC had a higher adsorption capacity for arsenic than MT. PC is synthesized by PC synthase (PCS) with glutathione (GSH) as its substrate.

Previous studies have found that PC synthase from Shizosomyces pombe can be expressed in SpPCS in E. coli and thus enhance the adsorption ability of E. coli to arsenic.

Therefore, we artificially synthesized SpPCS genes (partial base changes to make them conform to RFC10 standard) and expressed them in pET expression system.

We hope that the synergistic effect can be achieved by constructing the recombinant bacteria with adsorption within the bacteria and the adsorption using surface display, and thus we can remove arsenic ions from water more effectively.

In conclusion, we intend to build a biosensor to monitor the arsenic concentration in water and use the bioremediation device we built to remove arsenic ions in water.

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.The next day, 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 SpPCS fusion protein was 46Kda. 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.

Result

Under the induction of IPTG, the SpPCS protein was successfully expressed with a molecular weight between 40 KDa and 55 KDa, which was consistent with the predicted molecular weight of 46.7 KDa.

Fig.1. (a)IPTG-induced protein expression of recombinant plasmid pET42a-SpPCS in SDS-PAGE M：protein marker, 1: IPTG(-), 2: IPTG (1mM), 37℃,6h, 3: (1mM), 25℃,overnight. (b) IPTG-induced protein expression of recombinant plasmid pET42a-SpPCS in Western blot by using anti-Histag antibody.

As shown in figure 2, compared with the control group, the recombinant strain expressing SpPCS had a significantly higher adsorption capacity for arsenic than the control group. After adding 10μM of arsenic solution for 15 minutes, cells expressing SpPCS accumulated levels of arsenite was 1.73-fold higher than those accumulated by the control. As time increased to 45min, the adsorption capacity increased to 4.08-fold compared to the control, indicating that the SpPCS produced phytochelatin(PC) and successfully sequestered arsenic in the cells.

Fig.2 Arsenic accumulation in E. coli cells BL21(DE3) strain harboring the control vector(control) or expression SpPCS. The plasmid containing pET-SpPCS was transformed into BL21(DE3) and the next day a single colony was chose to culture overnight.Then the overnight culture was inoculated into the fresh LB broth at 1:100 , OD600 was 0.6-0.8. IPTG(1mM) was added and cultured at 28℃overnight. The bacteria was centrifuged and washed with PBS and resuspended into PBS. A final concentration of 10μM arsenite solution was added into the bacteria. Samples were taken every 15min, and 1ml was centrifuged at 5000rpm for 5min. The supernatant was collected and reserved for the detection of arsenic concentration. The arsenic in the samples were determined by the atomic fluorescence spectrometry.

Summary

the protein of the biosorption device we constructed has been successfully expressed. Compared with the control group, the recombinant bacteria expressing SpPCS significantly increased the adsorption capacity of arsenic.

In this project, based on the arsenic response part J33201, we have successfully established a biosensor that can quickly and sensitively detect arsenite(As3+) in water. Moreover, our arsenic biosorption can quickly and effectively remove arsenic from water.