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.It is notable that RBS B0030, a weaker ribosome binding site was used as to avoid the over expression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. 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

We mainly used two methods to evaluate the capability and efficacy of our bioabsorbent:

For qualitative measurement, we invented a creative method to directly visualize the detoxification process of our bioabsorbent. Firstly, we synthesized a water-soluble metal indicator, TritonX-100-PAN-S, according to the method in reference. Treated with pure sulfuric acid by stirring reaction overnight at room temperature, 1-(2-Pyridylazo)-2-naphthol (PAN) was sulfonated to make it water-soluble. (Scheme 2-1) After ether sedimentation and washing, we obtained the raw product, PAN-S, which was a reddish-brown solid powder. (Figure 2-2A) Then we mixed PAN-S and TritonX-100 by 1:20 mass ratio, and added ddH2O for thorough dissolving to get the final working solution, TritonX-100-PAN-S (TPS), with bright orange color. (Figure 2-2B)

Secondly, we conducted the characterization for our self-synthesized water-soluble metal indicator with expected colorimetric selectivity for heavy metals. We started with pH and metal concentration titration. By adding TPS into different pH solution at different mercury concentration, we found the proper color transition point at pH=~8. The lower limit of metal concentration for color transition was 0.8×10-5M. The color of solution changed from bright yellow to rosy color. (Figure 2-3)

Thirdly, we used our characterized indicator for direct visualization of mercury detoxification process. We constructed an integrated plasmid consisting of a constitutive promoter and metal binding peptides localized at different cell compartments. After transformation and overnight culturing, the bacteria were used to treat sample solution with 10-5M mercury and TPS at 37℃ for 10 min. The result was intriguing. Evident distinction could be observed between experiment group and control groups, which indicated the high efficacy of our bioabsorbent. (Figure 2-4)

For quantitative evaluation, we used inductively coupled plasma atomic emission spectrometry (ICP-AES) to precisely measure the different concentration of heavy metal before and after the treatment of bioabsorbent.

We constructed several plasmids for different localization of metal binding peptides and transformed them into BL21 (DE3) for expression, respectively. After approximately 40 hours of induction at the presence of different concentration of heavy metals, we collected the bacteria, washed with ddH2O for several times, and freeze-dried the pellet overnight before precise measuring their dry weights. Then we performed microwave digestion to completely dissolve all metal ions into solution before ICP-AES measurements. (Figure 2-5A, B)

The results are shown in the following figures: 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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