Latest revision as of 01:45, 21 October 2020

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 intend to build a bisabsorbent to remove arsenic ions in water.

Fig.1 Schemetic diagram of SpPCS protein expression in pET system

Fig.2 Genetic circuit of SpPCS 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.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.3. (a)IPTG-induced protein expression of recombinant plasmid pET42a-SpPCS in SDS-PAGE M：protein marker, 1: IPTG(-), 2: IPTG (1mM), 37℃,6h, 3: IPTG(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 4, 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.4 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.

Worldshaper-Wuhan 2020’s Contribution

Objective

Part K3153001 is a part constructed by the iGEM Worldshaper-Wuhan team in 2019. The result showed that this part can absorb arsenic in water. This year, we’d like to give future iGEM teams more information about this part so that everyone can understand it better and may use it in future projects.

Based on K3153001, a biosensor pET 24a-SpPCS was constructed to detect the change of arsenic adsorption rate over time. Since the absorption ability is affected by time, we increase the number of time points for testing the absorptivity to find out the relationship between absorption capacity and time.

Methods:

Plasmid pET24(a) and constructed biosensor pET 24(a)-SpPCs were 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 washed with PBS for 3 times. A final concentration of 5uM arsentic solution was added to the bacteria. Samples were taken at 5 time points, which were 5min, 15min, 30min, 45min, and 60min after adding the arsenic solution, respectively. The concentration of arsenic ions was tested by ICP-MS.

Result

As shown in the figure 1, A final concentration of 5uM arsentic solution was added to the bacteria. Samples were taken at 5 time points, which were 5min, 15min, 30min, 45min, and 60min after adding the arsenic solution, respectively. 5 minutes after adding 5uM arsenic solution, the arsenic absorption rate of the control group was 13%. At 15min, 30min, 45min and 60 min, the absorption rate of the control group was 15%, 14%, 17% and 17%, respectively. This number remained basically unchanged over time. On the other hand, at 5 minutes, the recombinant strain with SpPCs protein absorbed 14% of the arsenic, which showed no significant change compared with the control group. However, the absorption rate increased as time went on. When the arsenic solution was added for 30minutes, the absorption rate of arsenic reached 55%. After 30 minutes the increase in the absorption rate slowed down slightly. Even so, the absorption rate was a staggering 71% at 60 minutes, much higher than the17% of the control group.

Fig.1 Arsenic accumulation in E. coli cells

Conclusion

1. Compared with the control group, the recombinant strain expressing sppcs has a significantly higher adsorption capacity for arsenic than the control group, which is consistent with the initial results of this part. 2. Furthermore, according to our experimental results, with the increase of time: a. For control group, the arsenic adsorption rate is basically unchanged; b. For the recombinant bacteria, in the first 30 minutes, the adsorption rate increases rapidly, but the increase gradually slows down and tends to be saturated in the next 30minutes.

https://parts.igem.org/Part:BBa_K3153001