Part:BBa_K1355003:Experience

Experiments and Results

The experiment for Hg bioaccumulation was made according to the protocol “Quantification of Mercury bio accumulated by metal binding peptide (MBP) in recombinant DH5-alpha in different Hg concentrations”. DH5-alpha transformed with BBa_K1355003 was inoculated in LM (LB with low concentration of NaCl) liquid medium with chloramphenicol and grew until the optical density was 0.4 to 0.6abs (measured on spectrophotometer at 600 nm wavelength). After cell growth, an aliquot of 400μl was taken and distributed in 4 eppendorf tubes (1.5ml) and then added mercury chloride in order to achieve the concentrations: 50 ppb, 100 ppb, 250 ppb and 500 ppb. The samples were incubated at 37°C on shaker. We collected each eppendorf tube at time 1 (01:30 hours of incubation) and time 2 (03:00 hours of incubation). After the designated time, both were centrifuged at 12000g for 3 minutes and the supernatant recovered (LM medium). We washed the pellet with TN Buffer (Nacl 0.15M + Tris HCl 10mM) and then re-suspended with 400μl of the same buffer. To measure bio accumulated Hg, we need to quantify the Hg inside and outside of bacterium after the incubation/exposure time. So we collected and measured the amount of Hg in LM medium supernatant recovered and bacterium re-suspended in TN Buffer. For this we used the equipment Direct Mercury Analyzer (DMA-80). As a control to normal Hg bio accumulated in bacteria, we used DH5-alpha transformed with BBa_K1355002 (Hg bio detector device) which does not present the metal binding peptide. We also measured the Optical Density of each sample. The graph represented on Figure 1 shows the amount of Hg in supernatant (LM medium recovered) and in bacterium (DH5-alpha transformed with BBa_K1355003) at the time 1 (01:30 hours of incubation);

Figure 1: Metal binding peptide activity after 01:30 hours of incubation in five given concentrations of mercury chloride: 50 ppb, 100 ppb, 250 ppb e 500 ppb

It can be observed that the amount of Hg in the Mercury Bacter bioaccumulator increases according to the raise of Hg concentration. The amount of Hg increased 22 times comparing the 50 ppb sample with 500 ppb sample. In the 500 ppb Hg concentration the control bacterium just accumulated 2 per cent of total Hg amount. On the contrary, Mercury Bacter accumulated 40 per cent of total Hg amount in just 01:30 hours of incubation!!! The data keeps raising on the time 2! Check it out!

The graph represented on Figure 2 shows the amount of Hg in supernatant (LM medium recovered) and in bacterium (DH5-alpha transformed with BBa_K1355003) at the time 2 (03:00 hours of incubation);

Figure 2: Metal binding peptide activity after 03:00 hours of incubation in five given concentrations of mercury chloride: 50 ppb, 100 ppb, 250 ppb e 500 ppb.

It can be observed that the amount of Hg in Mercury Bacter bioaccumulator, increases according to the time of incubation. The amount of Hg increased 30 times comparing the 50 ppb sample with 500 ppb sample. In the 500 ppb Hg concentration the control bacterium just accumulated 4% of total Hg amount. Instead, Mercury Bacter accumulated 58% of total Hg amount in just 03:00 hours of incubation!!! The data can be analyzed individually in each concentrations samples as shown in graphs represented in the figures below:

Figure 3: Metal binding peptide activity at 50 ppb in time 1 and 2;

Figure 4: Metal binding peptide activity at 100 ppb in time 1 and 2;

Figure 5: Metal binding peptide activity at 250 ppb in time 1 and 2;

Figure 6: Metal binding peptide activity at 500 ppb in time 1 and 2

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