Part:BBa_K3153001( Deleted
Contents
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.
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.
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