Part:BBa_K346005:Experience

Experiment:

All parts of our bioabsorbent are driven by T7 promoter, followed by an RBS in the upstream of the part. T7 polymerase is constitutively expressed with the constitutive promoter Ptet. To avoid leakage and interference between different parts, a strong terminator of BBa_B0015 is added to the downstream of each part. It is necessary to notice that DsbA-mbp has to be located on the upstream of other parts, for there is a PstI enzyme digestion site, resulting in the only way to digest DsbA-mbp with EcoRI and SpeI. To test the function of the device, both expression experiment and function test is necessary. As a result,the size of the expressed proteins have been verified with SDS-page and Western blot. Besides, to verify the efficiency of mercury binding, we also carried out the function test with ICP-AES, which can test the quantity of mercury binding by the bacteria with the device.

Results:

Expression of proteins

The Dsba-mbp, mbp and lpp-ompa-mbp are inserted into the commercial plasmid PET21A. Then the plasmid is transferred to E.coli strain BL21, which can generate T7polyerase when induced with IPTG. Both induced cells and uninduced cells(as control) are centrifuged to get the cytosol, the periplasm and the membrane separated. The SDS-page and Western blot of the expressed proteins in these three parts(figure2) show that induced cells expressed an identical IPTG-inducible protein at the proper place with the size of ~12kD for MBP, ~40kD for DsbA-MBP and ~27kD for LPP-OMPA-MBP, all of which are consist with the predicted size, indicating that all these three coding sequence can be expressed normally in the right place.

Figure 1: The result of SDS-PAGE and Western blotting, indicating the expression of MBP, DsbA-MBP, lpp-OmpA-MBP in different location.

Function test

Having made sure that the protein can express normally in the proper place, the function tests experiment are carried out with ICP-AES. To test the efficiency of mercury absorption of our mercury bioabsorption device in different concentration of mercury, the concentration gradient is set from 10^-7M to 10^-5M, the results are shown in figure 3. It is obvious that the efficiency of this mercury absorption device increases with the increase of the mercury concentration and it can binding Hg(II) with high efficiency and high sensitivity from the concentration of 10^-6 M compared to that of the control. In addition, to compare the capacity of metal binding of the device which contains three subparts with the subparts alone(MBP, DsbA-MBP and Lpp-OMPA-MBP), these four parts are tested with the mercury concentration of 10^-5M to compare with each other, with the results shown in figure 4. It is necessary to point that that the device consisting of the three subparts seems to be less efficient than that of the surface display part Lpp-OmpA-MBP but it is better than the MBP in the cytoplasm and DsbA-MBP in the periplasm. This “unusual” phenomenon can be explained as that with the number of exogenous protein increases, the efficiency of expression of protein decreases quickly, for the hard burden due to these proteins.

Figure 3 && Figure 4The figure on the left showed the mercury binding capacity in different mercury concentration. We can see that the bacteria can absorbed more mercury in higher mercury concentration. But the bacteria had no significant mercury binding capacity when cultured with 0.1 uM mercury. But it is the detection limit of our biosensor. The figure on the right showed the mercury binding capacity of bacteria expressing differently localized MBPs. The surface displayed MBPs appear to have highest binding capacity while the pyramiding of MBP expression does not work well. This is because it is hard for the hydrophilic mercury ions to pass through the hydrophobic inner membrane.

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