Results
Biosensor
1. Construction of plasmids with lead-responsive biosensor
1) Preparation of vector and inserts
The schematic diagram of plasmid construction is shown below Fig.1a. The inserted fragments including the vector and inserts were obtained by enzyme digestion of our synthesize gene plasmid containing the target genes. As shown in Fig.1b and c, the length of vector, insert 1 (PbrR-S/M/W) and insert 2 (pbrA-mCherry) was about 2000bp, 600bp and 1000bp respectively, as expected.
Fig.1.a
Fig.1.Construction of lead-responsive plasmid. (a) schematic diagram of plasmid construction. (b) enzyme digestion of plasmid, Lane M,DL5000 marker, Lane1, pSB1C3PbrR-M(E/S), Lane2, pSB1C3PbrA-mCherry(X/P), Lane 3, pSB1C3PbrR-M (E/P),Lane 4, pSB1C3PbrR-M (E/S),Lane 5, pSB1C3PbrR-W (E/S). (c) gel extraction of enzyme digestion product for vector and inserts, Lane M, DL5000 marker, Lane1, PbrR-W, lane2: PbrR-M, lane3, PbrR-S, lane 4, mCherry, lane 5, Vector.
2) Ligation and transformation
The purified inserted fragments and vector were ligated by T4 ligase, and the ligation product was transformed into E. coli DH5α. As shown in Fig.2, the transformation of the ligation was successful. There was no colony growth on the carrier, but there was a lot of growth on the recombinant plate, indicating that the ligation is successful.
Fig. 2 Results of transformation for ligated products: growth of plate colony. a, vector control; b, pSB1C3-PbrR-S-pbrA-mCherry (S) c, pSB1C3-PbrR-M-pbrA-mCherry(M); d,pSB1C3-PbrR-W-pbrA-mCherry(W).
3) Identification of bacterial recombinants
As shown in Fig.3a, five positive clones were selected from the plate for bacteria colony PCR. The results showed that there were many positive clones. The extracted plasmid was identified by enzyme digestion, and the constructed plasmid was digested by EcoR I/Pst I double enzyme digestion, and we selected two of the five plasmids to identify by enzyme digestion, the enzyme digestion profiles of the recombinant plasmid were in line with expectations (figure 3b).
Fig.3. (a) Bacterial colony PCR product of recombinants in gel electrophoresis. Lane M, Marker. Lane S1-S5, pSB1C3-PbrR-S-pbrA-mcherry, Lane M1-M5, pSB1C3-PbrR-M-pbrA-mcherry, W1-W5, pSB1C3-PbrR-W-pbrA-mcherry, positive clones of recombinant plasmid. (b) Profiles of enzyme digestion of recombinant plasmid (EcoR I/Pst I) .Lane M, Marker, Lane 1-3, pSB1C3-PbrR-S-pbrA-mcherry, Lane 4-6, pSB1C3-PbrR-M-pbrA-mcherry, Lane 7-9 pSB1C3-PbrR-M-pbrA-mcherry.
4). DNA sequencing
Sequencing results showed that the sequence was completely correct and in line with expectations. Some of the plasmids were sent to the company for sequencing, and S4, M5 and W1 were selected as recombinant plasmids and recombinant bacteria for the following experiment.
To summary, the plasmid construction of our biosensor is successful.
2. The function of the constructed biosensors
1) Growth curve of different concentrations of lead to recombinant bacteria
As shown in Fig. 4, effects of different concentrations of lead on the growth of three biosensor, pSB1C3-PbrR-S-pbrA-mCherry (S), pSB1C3-PbrR-S-pbrA-mCherry (M), pSB1C3-PbrR-S-pbrA-mCherry( W) were investigated. The growth of S strain was not affected within the range of the given lead concentration from 10ppb to 10ppm(Fig.4a), while in the M strain, the growth was slightly inhibited at 5ppm and 10ppm of lead(Fig.4b). In contrast, W stain was less resistant to lead when compared with strains of S and W, the bacterial growth was strongly inhibited in the high concentration of lead at at 5ppm and 10ppm (Fig.4c).
The results of the growth curve indicated that the PbrR protein adsorbed lead,which resulted in the resistance of recombinant bacteria to lead.
Fig.4a.
Fig.4b.
Fig.4c.
Fig.5 Growth curve of E.coli in different concentration of lead (Pb2+) under the control of different strength of PbrR S (a), M(b) ,W(c). Constructed plasmid S, M,W part was transformed into E.coli DH5α strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in the fresh LB medium containing chloramphenicol 34μg/ml at a ratio of 1:100, mixed well and divide into tubes. Different concentrations of lead (Pb2+) solutions were added into the test tubes, respectively, so that the final concentration was (0, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm). Samples were taken at different time points of 0h, 1h, 2h…6h and overnight (16h) at one hour interval and OD600 value were measured with a Multiskan Spectrum Microplate Reader.
2) RFP expression of different concentrations of Pb2+ to recombinant bacteria
As shown in Fig.5, RFP expression was not significantly different under different lead concentrations, suggesting that biosensor constructed by recombinant plasmid did not work as expected. This result has been repeated many times, but no expected fluorescence signal has been detected, indicating that the biosensor based on lead-responsive promoter pPbrA is invalid. As a result, it was suspended due to no function.
Fig. 5
Fig.5 RFP expression of E.coli in different concentration of lead (Pb2+) at different time points under the control of PbrR and pbrA promoter. Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol resistance at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of lead solutions were added into the test tube, respectively, so that the final concentration was (0, 5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm, 100ppm). Samples were taken overnight (16h) and RFP fluorescence intensity (585 nm excitation/ 610 nm emission) and OD600 value were measured at the same time
3) PbrR expression of under the control of different strength of Anderson promoters
According to Fig 6, PbrR protein, a lead binding protein, was expressed under different strength of constitution titutive promoters, J23100(S), J23106(M) and J23117(W). The order is S>M>W. This also explains why the Biosensor constructed in sequence, S,M and W have different tolerance to lead. As shown in Fig7, biosensor containing PbrR-S has a greater resistance to Pb2+, while biosensors composed of PbrR-M or PbrR-W have a poor tolerance to higher concentration (5ppm and 10ppm) of lead, indicating that PbrR expression plays a certain role in the adsorption of lead in the cell.
Fig6. PbrR protein expression under different strength of Anderson promoters in SDS-PAGE. S, strong Anderson promoter J23100, M, medium promoter, J23106, W, weak promoter J23117,
Fig7. Effect of PbrR protein expression under the control of different strength of promoters on bacteria resistance to lead.
Taken together, we have successfully constructed the plasmids for biosensor. It was also found that PbrR protein was successfully expressed and expressed differently according to the strength of promoter, resulting in different biosensor tolerance to lead. However, the biosensor constructed by us did not work as expected.