Capture part
1.1 SDS-PAGE verification for oprF-3*LBT 4 & oprF-Sitag expression
Whether the oprF-3*LBT or oprF-Sitag can be expressed or not needs to be detected by SDS-PAGE.
Firstly, we need to identify that OprF-3* LBT4 and OprF-Sitag are expressed on the surface of the E. coli. We centrifugated the bacteria solution and got the whole test BL21 cells induced by IPTG. As control, we also extracted the not induced BL21 cells. Then we separate the protein of the whole cell by SDS-PAGE.
Figure 1 shows an obvious ~33kDa protein bands in test lane, which cannot be found in the control lane. This result proves that the test BL21 can produce OprF-3* LBT 4.
Figure 2 shows an obvious ~57kDa protein bands in test lane, which cannot be found in the not induced lane. This result proves that the test BL21 can produce OprF-Sitag.
1.2 Verification of Cell Surface Display System by Immunofluorescent assay
Then we did immunofluorescence assay to figure out whether the cell surface display system works or not.
Figure 3 shows the result of our verification of our cell surface display system. Some cells can show a considerable fluorescent intensity, while some perform partial or weak fluorescence which can not be detected by our microscope camera. But this result still successfully demonstrated the cell surface display of our LBTs.
1.3 Verification of Tb3+ capturing by precipitation titration of remained Tb3+
To measure REEBOT’s ability to capture Tb ions, one kind of lanthanide ions, we have done a precipitation titration of Tb hydroxide. We first add REEBOT into Tb ion solution. Each of them has certain concentration and volume. After incubation, we discard the bacteria, and then titrate the solution with sodium hydroxide. The less precipitation of Tb hydroxide generates, the more Tb ions are captured by the bacteria.
Figure 4 shows that it is significant difference in the amount of precipitation. The amount of precipitation in WT group(with distilled water added) is much less than that in the test group.
To get some more detailed and convincing results, we did a precipitation-dissolving titration with 0.1M hydrochloric acid. Figure 5 can show a result that the test group(with our engineered bacteria) is much different from the control group. After our engineered bacteria work a while in sample, the terbium ions concentration is significantly reduced.
