Section 1 : Introduction

Our project was designed to include identification of rare earth elements, adsorbtion and desorption rare earth elements by engineering E. coli and recycling rare earth elements. In order to achieve these goals, we used Escherichia coli to construct all kinds of plasmids we needed, and transferred them into E. coli BL21 for expressing the proteins we needed. At the same time, in order to verify the rationality of the recycle of rare earth elements, we also carried out various experiments on the recycle of rare earth elements using industrial wastewater, and verified our project from all aspects.

Section 2 : Plasmid construction and amplification

We first transformed the plasmids containing designed sequence synthesized by the company into Escherichia coli for amplification, so as to carry out subsequent experiments. Then colony PCR showed a successful transformation, and required plasmids were extracted for the next experiment.

Fig. 1. Colony PCR confirmation of successful E. coli transformation

We transformed the plasmids containing the target gene into Escherichia coli, and obtained a large number of target genes through culturing Escherichia coli, so as to carry out subsequent operations.

Fig. 2. The PCR results of (pmrC)oprf-sitag-LanM and PmrB(LBT-LanM)-PmrC

The bands of (pmrC)oprf-sitag-LanM(almost 2000bp) and pmrB(LBT-LanM)-PmrC(almost 2000bp) from PCR are identical to the theoretical lengths of 1797bp and 1735 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmids had successfully been transformed into E. coli.

Fig. 3.The PCR results of (pmrC)oprf-sitag-FP and pmrB-PmrC-oprf-sitag-FP

The bands of (pmrC)oprf-sitag-FP(2000+bp) and pmrB-PmrC-oprf-sitag-FP(2000+bp) from PCR are identical to the theoretical lengths of 2010p and 2311bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmids were successfully constructed.

Fig. 4. The PCR result of (PgolB)oprf-sitag-LanM

The band of (PgolB)oprf-sitag-LanM Terminator from PCR is almost 2000bp, identical to the theoretical length of 1863bp estimated by the designed primer location (promoter to terminator), which could demonstrate that this target plasmid had successfully been transformed into E. coli.

Fig. 5. The PCR result of (PgolB)oprf-sitag-FP

The band of (PgolB)oprf-sitag-FP from PCR is about 2000bp, identical to the theoretical length of 2013bp estimated by the designed primer location (promoter to terminator), which could demonstrate that this target plasmid had successfully been transformed into E. coli.

Fig. 6. Plasmid construction and PCR results of pmrB(LBT-LanM) and pmrB(LanM)

The bands of pmrB(LBT-LanM) (1000+bp) and pmrB(LanM) (1000+bp) from PCR are identical to the theoretical lengths of 1176p and 1100bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmids were successfully constructed.

Fig. 7. Plasmid construction and PCR results of pmrB（LanM）-PmrC and PmrC.

The bands of pmrB（LanM）-pmrC (almost 2000bp) and pmrC (almost 750bp) from PCR are identical to the theoretical lengths of 1795bp and 745bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmids were successfully constructed.

Fig. 8. Plasmid construction and PCR results of golS-T7 terminator-PgolB and T7-KanR

The bands of golS-PgolB (about 2500bp) and T7-KanR (3000+bp) from colony PCR are identical to the theoretical lengths of 2529bp and 3296p estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmids were successfully constructed.

Section 3 : Conclusion

We had successfully constructed all target plasmids we needed in our four circuits which could be demonstrated by the image of PCR colony as follows. The bands of T7-pmrA-T7-pmrB(LanM)-T7 Terminator-PmrC-oprf-sitag-LanM-T7 Terminator (about 5000bp), T7-pmrA-T7-pmrB(FP)-T7 Terminator-PmrC-oprf-sitag-FP -T7 Terminator (about 5000bp), T7-golS-T7 terminator-PgolB-oprf-sitag-LanM-T7 Terminator (about 5000bp) and T7-golS-T7 terminator-PgolB-oprf-sitag-FP-T7 Terminator (about 5000bp) from colony PCR are identical to the theoretical lengths of 4473bp, 4686bp, 4392bp and 4542bp estimated by the designed primer locations (promoter to terminator).

This step really facilitated us to carry out subsequent experiments. After successful transformation was verified by colony PCR, the required plasmids were extracted for the next experiment.

Fig. 9. Plasmid construction and colony PCR results of T7-pmrA-T7-pmrB(LanM)-T7 Terminator-PmrC-oprf-sitag-LanM-T7 Terminator,T7-pmrA-T7-pmrB(FP)-T7 Terminator-PmrC-oprf-sitag-FP -T7 Terminator,T7-golS-T7 terminator-PgolB-oprf-sitag-LanM-T7 Terminator,T7-golS-T7 terminator-PgolB-oprf-sitag-FP-T7 Terminator transformed E.coli

The bands of T7-pmrA-T7-pmrB(LanM)-T7 Terminator-PmrC-oprf-sitag-LanM-T7 Terminator (about 5000bp), T7-pmrA-T7-pmrB(FP)-T7 Terminator-PmrC-oprf-sitag-FP -T7 Terminator (about 5000bp), T7-golS-T7 terminator-PgolB-oprf-sitag-LanM-T7 Terminator (about 5000bp) and T7-golS-T7 terminator-PgolB-oprf-sitag-FP-T7 Terminator (about 5000bp) from colony PCR were identical to the theoretical lengths of 4473bp, 4686bp, 4392bp and 4542bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmids were successfully constructed.

Section 4 : Expression Validation

After we constructed the plasmids corresponding to PmrL, PmrFP, GolSL, GolSFP system pathways successfully, we transformed the constructed plasmids into E. coli BL21 and used colony PCR to verify that our plasmids were transferred successfully for the experiments of expression validation.

Fig. 10. The PCR of PmrL, PmrFP, GolSL and GolSFP colonies all had bright and correct bands, proving that the transformation was successful

4.1 Validation of induction protein expression

For the induction protein expression validation, we extracted the induction proteins and verified them by SDS-PAGE, and we successfully observed the target bands on the SDS-PAGE gel and showed a successful expression.

Fig. 11. After purifying PmrL by nickel column, SDS-PAGE verification showed that there was a band at 25-30kDa which was significantly different from that of throughflow and impurity washing, which confirmed that PmrA was expressed in cells

Fig. 12. After purifying GolSFP by nickel column, SDS-PAGE verification showed that there was a band at 15-20 kDa that were significantly different from that of throughflow and impurity washing, which confirmed that GolS was expressed in cells

Fig. 13. After PmrL induction and culture, membrane protein was extracted for SDS-PAGE verification. Compared with the control group without induction, there were clear bands at about 38kDa showing PmrB successful expression

4.2Validation of capture protein expression

For the capture protein expression validation, we not only verified the expression of the proteins, but also tested their function and location. Finally we got some success.

4.2.1 Verification of membrane protein expression

For the PmrL and PmrFP pathways, they can induce the REEs and then express the capture proteins. To test the function of the induction protein and verify the expression of the capture proteins, we added different REEs respectively into the culture to induce and extracted membrane proteins to verify them by SDS-PAGE. After some attempts, the target bands were observed on the gel, and we confirmed that our engineering bacteria can identify REEs before expressing capture proteins. Besides, we found that the efficiency of induction was optimal when using Tb³⁺ to induce.

Fig. 14. The left figure shows that PmrL was induced by Tb, Sc, Ce, La, Yb respectively and SDS-PAGE result of each membrane proteins. It can be seen that there are clear bands at 63kDa, which can verify its successful expression. The right figure shows that PmrFP was induced by Tb, Sc, La and Yb respectively and SDS-PAGE result of each membrane proteins. It can be seen that there are clear bands at 63kDa, which can verify its successful expression.

For the GolSL and GolSFP system pathways, we extracted membrane proteins after induction and observed the target band at 63 kDa which proved that our capture proteins expressed successfully.

Fig. 15. The left figure shows the membrane protein of GolSL extracted after induction for SDS-PAGE verification. Compared with the control group without induction, it can be seen that there are clear bands at 63kDa to judge its successful expression; The right figure shows the membrane protein of GolSFP extracted after induction for SDS-PAGE verification. Compared with the control group without induction, it can be seen that there are clear bands at 63kDa to judge its successful expression.

4.2.2 Verification of membrane protein location

We tried to use immunofluorescence assay to verify that the membrane proteins located on the cell membrane. We added a His-tag to the C-terminus of the fusion protein when we designed the system pathways so that we can use immunofluorescence labelling. Finally, We successfully observed the engineering bacteria emitting fluorescence by fluorescence microscope and confirmed that the membrane protein was indeed located at the right position.

Fig. 16. The figure of PmrL after immunofluorescence assay treated under fluorescence microscope. The E. coli emitting green fluorescence could be observed which can confirm he membrane protein located at the right position.

Fig. 17. The figure of GolSFP after immunofluorescence assay treated under fluorescence microscope. The E. coli emitting green fluorescence could be observed which can confirm the membrane protein located at the right position.

4.2.3 Verification of Si-tag adsorbtion

We tried to test the Si-tag adsorbtion capacity of engineered bacteria by adding the bacteria solution to the silica column filled with coarse sand which have been balanced with Tris buffer and incubated it for 6h. During this period, we took 2ml of the effluent every hour to measure its OD₆₀₀ value, so as to obtain the change of the concentration of the bacterial solution and verify the ability of Si-tag adsorbtion. After the 6h of incubation, the adsorbtion rate of engineering bacteria is up to 89%.

Fig. 18. OD value of PmrL absorbed by coarse sand. The red is the control group, while the black is PmrL. It can be seen that the OD value decreased significantly after 6 hours, which can prove that the adsorbtion capacity of Si-tag is very high.

Besides, we used field emission scanning electron microscope to observe the quartz sand samples after the adsorbtion experiment, and we could observe the bacteria binding to the silica.

Fig. 19. A: The SEM image of E. coli cells in the experimental group adhered on the surface of silica particle. B: The SEM image of the control group.

4.2.4 Verification of adsorbtion capacity of rare earth elements

To test the REEs adsorbtion ability of engineering bacteria, we constructed the experimental system of absorbing rare earth metal ions. Subsequently, we added an appropriate concentration of rare earth ions to the adsorbtion system and incubated at 100rpm,30℃ for 2h before centrifugation. Then, we determined the concentration of rare earth ions in the supernatant by ICP-OES After several attempts, the engineered bacteria of our PmrL system pathway got an optimal outcome—the averaged Tb³⁺ adsorbtion efficiency was 84%.

Fig. 20. The Tb³⁺ adsorbtion of PmrL, PmrFP, GolSL, GolSFP

After verifying the engineered bacteria ability of absorbing REEs, we tried to add different kinds of REEs to test the adsorbtion rate of our target proteins in different REEs.

Fig. 21. The efficiency of PmrL absorbing La, Ce, Sm, Nd, Eu, Y, Gd, Sc

In addition, we added engineering bacteria into industrial wastewater rich in REEs and used the same way to detect the efficiency. Finally, the Sm³⁺ averaged adsorbtion of PmrL in pH=5.22 reached 93%

Fig. 22. Different REEs adsorbtion of PmrL system in industrial wastewater rich in REEs with different pH

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