Section 1 : Molecular Experiment

6.30-7.1

Transform plasmids (T7-pmrA-PmrC of Pmr-LanM pathway, T7-pmrA-PmrC of Pmr-dLBT-LanM pathway) to E. coli TOP10F’.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

Inoculate E. coli T7-pmrA-PmrC of Pmr-LanM pathway, T7-pmrA-PmrC of Pmr-dLBT-LanM pathway.

7.2

Extract plasmids（T7-pmrA-PmrC of Pmr-LanM pathway, T7-pmrA-PmrC of Pmr-dLBT-LanM pathway）.

PCR and electrophorese to confirm.

Fig. 1. The left figure is the PCR identification result of pmrA-T7-Terminator The middle figure is is the PCR identification result of T7-pmrA-PmrC of PmrFP The right figure is the PCR identification result of PmrC

Purification of T7-pmrA of Pmr-LanM pathway, T7-pmrA of Pmr-dLBT-LanM pathway, PmrC.

7.3-7.4

Transform plasmids (pmrB (LanM), pmrB (LBT-LanM)) to E. coli TOP10F’.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Combined with water and glycerol, preserve.

Inoculate E. coli pmrB (LanM), pmrB (LBT-LanM).

7.5-7.6

Extract plasmids（pmrB (LanM), pmrB (LBT-LanM)）.

PCR and electrophorese to confirm.

Fig. 2. The left figure is the PCR identification result of pmrB(LanM) The right figure is the PCR identification result of pmrB(LBT-LanM)

Transform plasmids (oprf-sitag-LanM, oprf-sitag-dLBT-LanM) to E. coli TOP10F’.

7.7-7.9

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Combined with water and glycerol, preserve.

Inoculate E. coli oprf-sitag-LanM, oprf-sitag-dLBT-LanM.

7.10

Extract plasmids (oprf-sitag-LanM, oprf-sitag-dLBT-LanM).

PCR and electrophorese to confirm.

Fig. 3. The left figure is the PCR identification result of oprF-sitag-LanM of PmrL The right figure is the PCR identification result of oprF-sitag-dLBT-LanM of PmrFP

Purification of oprf-sitag-LanM, oprf-sitag-dLBT-LanM.

Homologous recombination of Pmr-LanM by T7-pmrA-T7 Terminator of Pmr-LanM pathway, pmrB (LanM), PmrC, oprf-sitag-LanM.

Transform the plasmids (homologous recombination) to E. coli TOP10F’.

7.11

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

We didn’t succeed in connecting the four fragments and planned to try SOE PCR.

7.12

Transform plasmids (T7-T7 Terminator of GolS-LanM pathway, T7-T7 Terminator of GolS-dLBT-LanM pathway) to E. coli TOP10F’.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

Inoculate E. coli T7-T7 Terminator of GolS-LanM pathway, T7-T7 Terminator of GolS-dLBT-LanM pathway.

7.13

Extract plasmids（T7-T7 Terminator of GolS-LanM pathway, T7-T7 Terminator of GolS-dLBT-LanM pathway）.

PCR and electrophorese to confirm.

Fig. 4. The left figure is the PCR identification result of T7-T7 Terminator of GolSL The right figure is the PCR identification result of T7-T7 Terminator of GolSFP

Purification of T7-T7 Terminator of GolS-LanM pathway, T7-T7 Terminator of GolS-dLBT-LanM pathway.

7.14

Transform plasmids (GolS-PgolB) to E. coli TOP10F’.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm

Combined with water and glycerol, preserve.

Inoculate E. coli GolS-PgolB.

7.15

Extract plasmids（GolS-PgolB）.

PCR and electrophorese to confirm.

Fig. 5. The PCR identification result of golS-PgolB

Purification of GolS-PgolB.

Transform plasmids (oprf-sitag-LanM of GolS-LanM pathway, oprf-sitag-dLBT-LanM of GolS-dLBT-LanM pathway) to E. coli TOP10F’.

7.16-7.17

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Combined with water and glycerol, preserve.

Inoculate oprf-sitag-LanM of GolS-LanM pathway, oprf-sitag-dLBT-LanM of GolS-dLBT-LanM pathway.

Extract plasmids（oprf-sitag-LanM of GolS-LanM pathway, oprf-sitag-dLBT-LanM of GolS-dLBT-LanM pathway）.

PCR and electrophorese to confirm.

Fig. 6. The left figure is the PCR identification result of oprF-sitag-LanM of GolSL The right figure is the PCR identification result of oprF-sitag-dLBT-LanM of GolSFP

Purification of oprf-sitag-LanM of GolS-LanM pathway, oprf-sitag-dLBT-LanM of GolS-dLBT-LanM pathway.

7.18

Homologous recombination of GolS-LanM by T7-T7 Terminator of GolS-LanM pathway, GolS-PgolB, oprf-sitag-LanM of GolS-LanM pathway.

Transform the plasmids (homologous recombination) to E. coli TOP10F’.

SOE PCR of pmrB(LanM) and PmrC and get pmrB (LanM)-T7 Terminator-PmrC by gel recycle.

Fig. 7. The PCR identification result of pmrB(LanM)-pmrC promoter of PmrL

7.19

SOE PCR of pmrB (LanM)-T7 Terminator-PmrC and oprf-sitag-LanM and get pmrB (LanM)-T7 Terminator-PmrC-oprf-sitag-LanM by gel recycle.

SOE PCR of pmrB (LBT-LanM) and PmrC and get pmrB (LBT-LanM)-T7 Terminator-PmrC by gel recycle.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Fig. 8. The left figure is the PCR identification result of pmrB(LanM)-T7 Terminator-pmrC promoter-oprF-Sitag-LanM The right figure is the PCR identification result of pmrB(LBT-LanM)-pmrC promoter

Fig. 9. The PCR identification result of golS (Cu²⁺)-T7 Terminator-PgolB-oprf-sitag-dLBT-LanM

We could see that homologous recombination brought a correct result and we got GolS-LanM plasmid.

7.20

SOE PCR of pmrB (LBT-LanM)-T7 Terminator-PmrC and oprf-sitag-dLBT-LanM and get pmrB (dLBT-LanM)-T7 Terminator-PmrC-oprf-sitag-dLBT-LanM by gel recycle.

Homologous recombination of Pmr-dLBT-LanM by T7-pmrA-T7 Terminator of Pmr-dLBT-LanM pathway, pmrB (LBT-LanM)-T7 Terminator-PmrC, oprf-sitag-dLBT-LanM.

Homologous recombination of GolS-dLBT-LanM by T7-T7 Terminator of GolS-dLBT-LanM pathway, GolS-PgolB, oprf-sitag-dLBT-LanM of GolS-dLBT-LanM pathway.

Transform the plasmids (homologous recombination) to E. coli TOP10F’.

7.21

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Homologous recombination of Pmr-dLBT-LanM by T7-pmrA-T7 Terminator of Pmr-dLBT-LanM pathway, pmrB (LBT-LanM)-T7 Terminator-PmrC-oprf-sitag-dLBT-LanM.

Homologous recombination of Pmr-LanM by T7-pmrA-T7 Terminator of Pmr-LanM pathway, pmrB (LanM)-T7 Terminator-PmrC-oprf-sitag-LanM.

Transform the plasmids (homologous recombination) to E. coli TOP10F’.

7.22

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Fig. 10. The left figure is the PCR identification result of T7-pmrA- pmrB(LanM)-T7 Terminator-pmrC promoter-oprF-sitag-LanM-T7 Terminator The right figure is the PCR identification result of T7-pmrA- pmrB(LBT-LanM)-T7 Terminator-pmrC promoter-oprF-sitag-dLBT-LanM-T7 Terminator

Through homologous recombination, we constructed four pathway’s expression vector: Pmr-LanM, Pmr-dLBT-LanM, GolS-LanM, GolS-dLBT-LanM. Then we would transformed the plasmids into E. coli BL21 to express.

7.23-7.24

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM, GolS-LanM, GolS-dLBT-LanM.

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM, GolS-LanM, GolS-dLBT-LanM).

Transform the plasmids to E. coli TOP10F’.

7.25

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Combined with water and glycerol, preserve.

7.26

Homologous recombination of Pmr-dLBT-LanM by T7-pmrA-T7 Terminator of Pmr-dLBT-LanM pathway, pmrB (LBT-LanM)-T7 Terminator-PmrC-oprf-sitag-dLBT-LanM.

Transform the plasmids (homologous recombination) to E. coli TOP10F’.

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM.

Transform plasmids (GolS-LanM) to E. coli BL21.

7.27

SOE PCR of GolS-PgolB and oprf-sitag-LanM of GolS-LanM pathway, GolS-PgolB and oprf-sitag-dLBT-LanM of GolS-dLBT-LanM pathway，gel recycle.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Combined with water and glycerol, preserve.

Transform plasmid (Pmr-LanM) to E. coli BL21

Transform plasmid (Pmr-dLBT-LanM) to E. coli TOP10F’.

7.28

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Combined with water and glycerol, preserve.

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM.

7.29

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM) and PCR to confirm.

Transform plasmid (Pmr-LanM, Pmr-dLBT-LanM) to E. coli BL21.

7.30

Pick the bacterial plaque (Pmr-LanM, BL21). PCR bacterial fluid, and electrophorese to confirm.

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM.

7.31

Pick the bacterial plaque (Pmr-dLBT-LanM, BL21). PCR bacterial fluid, and electrophorese to confirm.

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM) and PCR to confirm.

8.1

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM.

Combined with water and glycerol, preserve Pmr-dLBT-LanM.

8.2

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM) and PCR to confirm.

Combined with water and glycerol, preserve Pmr-LanM.

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM.

8.3

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM) and PCR to confirm.

Combined with water and glycerol, preserve Pmr-dLBT-LanM.

Inoculate Pmr-LanM (BL21), Pmr-LanM (TOP10F’), Pmr-dLBT-LanM (TOP10F’).

PCR of pmrB (dLBT-LanM)-T7 Terminator-PmrC-oprf-sitag-dLBT-LanM, purification.

8.4

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM) and PCR to confirm.

Homologous recombination of Pmr-dLBT-LanM by T7-pmrA-T7 Terminator of Pmr-dLBT-LanM pathway, pmrB (dLBT-LanM)-T7 Terminator-PmrC-oprf-sitag-dLBT-LanM.

Homologous recombination of Pmr-LanM by T7-pmrA-T7 Terminator of Pmr-LanM pathway, pmrB (LanM)-T7 Terminator-PmrC-oprf-sitag-LanM.

Transform the plasmids (homologous recombination) to E. coli TOP10F’.

8.5

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Combined with water and glycerol, preserve Pmr-LanM, Pmr-dLBT-LanM.

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM.

8.6

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM) and PCR to confirm.

Transform the plasmids to E. coli BL21.

8.7

Transform the plasmids (Pmr-LanM) to E. coli BL21.

Homologous recombination of Pmr-dLBT-LanM by T7-pmrA-T7 Terminator of Pmr-dLBT-LanM pathway, pmrB (LBT-LanM)-T7 Terminator-PmrC-oprf-sitag-dLBT-LanM.

Transform the plasmids to E. coli TOP10F’.

8.8

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

SOE PCR of GolS-PgolB and oprf-sitag-dLBT-LanM of GolS-dLBT-LanM pathway and get golS (Cu²⁺)-Terminator-PgolB-oprf-sitag-dLBT-LanM by gel recycle.

Fig. 11. The PCR identification result of golS (Cu²⁺)-T7 Terminator-PgolB-oprf-sitag-dLBT-LanM

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM.

8.9

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM) and PCR to confirm.

Homologous recombination of GolS-dLBT-LanM by T7-T7 terminator of GolS-dLBT-LanM pathway and golS (Cu)-Terminator-PgolB-oprf-sitag-dLBT-LanM.

Transform the plasmids to E. coli TOP10F’.

Single enzyme digestion of Pmr-LanM to confirm.

8.10

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

8.11

Transform the plasmids (GolS-dLBT-LanM) to E. coli TOP10.

8.12

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM.

8.13

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM) and PCR to confirm.

Fig. 12. The PCR identification result of T7-pmrA- T7-pmrB(LBT-LanM)-T7 Terminator- PmrC-oprf-sitag-dLBT-LanM-T7 Terminator

Transform the plasmids (GolS-LanM) to E. coli BL21.

Combined with water and glycerol, preserve GolS-dLBT-LanM.

8.14

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

Inoculate E. coli Pmr-LanM, Pmr-dLBT-LanM, GolS-LanM.

Transform the plasmids (GolS-dLBT-LanM) to E. coli BL21.

Combined with water and glycerol, preserve Pmr-LanM and GolS-LanM.

8.15

Pick the bacterial plaque (GolS-dLBT-LanM). PCR bacterial fluid, and electrophorese to confirm.

Inoculate Pmr-LanM, Pmr-dLBT-LanM, GolS-LanM, GolS-dLBT-LanM.

Combined with water and glycerol, preserve GolS-dLBT-LanM.

8.16-8.17

Extract plasmids (Pmr-LanM, Pmr-dLBT-LanM, , GolS-LanM, GolS-dLBT-LanM) and PCR to confirm.

Fig. 13. The left figure is the PCR identification result of T7-golS-T7 Terminator-PgolB- oprf-sitag-LanM- T7 Terminator The right figure is the PCR identification result of 7-golS-T7 Terminator- PgolB- oprf-sitag-FP- T7 Terminator

SOE PCR of pmrB (LBT-LanM) and PmrC promoter and get pmrB (LBT-LanM)-T7 Terminator-PmrC by gel recycle.

SOE PCR of pmrB (LBT-LanM)-T7 Terminator-PmrC and oprf-sitag-dLBT-LanM and get pmrB (dLBT-LanM)-T7 Terminator-PmrC-oprf-sitag-dLBT-LanM by gel recycle.

8.18-8.19

Homologous recombination of Pmr-dLBT-LanM by T7-pmrA-T7 Terminator of Pmr-dLBT-LanM pathway, pmrB (LBT-LanM)-T7 Terminator-PmrC-oprf-sitag-dLBT-LanM.

Transform the plasmids (homologous recombination) to E. coli TOP10F’.

8.20

Extract plasmids (Pmr-dLBT-LanM) and PCR to confirm.

Transform the plasmids (Pmr-dLBT-LanM) to E. coli BL21.

8.21-9.20

We kept inoculating Pmr-LanM, Pmr-dLBT-LanM, GolS-LanM, GolS-dLBT-LanM and inducing, and the E. coli BL21 were used to conduct expression experimen

Fig. 14. Colony PCR confirmation of successful E. coli transformation

Section 2 : Expression Experiment

8.5-8.28

We first tried confirming that the target proteins had been successfully expressed while anchored on the cell membrane. We inoculated E. coli BL21 with Pmr-LanM, Pmr-dLBT-LanM, GolS-LanM, GolS-dLBT-LanM and induced E. coli BL21 Pmr-LanM and Pmr-dLBT-LanM with IPTG and Tb³⁺, while inducing E. coli BL21 with GolS-LanM and GolS-dLBT-LanM with IPTG and Cu²⁺.

IPTG: 0.5 mM Tb³⁺: 50 µM Cu²⁺: 50 µM

We used the following method to confirm.

-Centrifugation of bacteria solution with 6000 rpm, then dissolving with PBS solution.

-Cell fragmentation by ultrasound, then centrifugation with 12000 rpm to get membrane protein precipitation.

-Treated the precipitation with TDSET buffer and SDS-PAGE to confirm.

After preliminary validation, we used a wider range of rare earth ions to induce E. coli BL21 with Pmr-LanM and Pmr-dLBT-LanM. We repeated the same method and also got correct results.

8.15-8.23

We used immunofluorescence assay to prove that the target proteins had been anchored on the cell membrane. We conducted two rounds of experiments to vertify.

We used the following method to prepare the samples.

-Centrifugation of bacteria solution with 12000 rpm, then washing with PBS solution twice.

-Dissolving with 50 µl PBS (with 1 mg/ml BSA).

-Adding His-tag primary antibody and incubation for 3 hours.

-Centrifugation of solution, then washing with PBS solution twice.

-Dissolving with 50 µl PBS (with 1 mg/ml BSA).

-Adding IgG（H+L）with FITC labeling second antibody and incubation for 3 hours.

-Centrifugation of solution, then washing with PBS solution twice and dissolving with PBS buffer.

-Using fluorescence microscope to observe.

The result showed that the fusion protein was indeed anchored on the cell membrane.

Fig. 15. PmrL

Fig. 16. PmrFP

Fig. 17. GolSL

Fig. 18. GolSFP

9.9-9.15

We built partnership with team Tsinghua and discussed about our experiments in many aspects. They sent their materials to us and we helped them to test the expression of their functional protein.

We inoculated their engineered E. coli but they failed to grow in our culture medium. We thought that the transport process resulted in decreasing of bacterial activity, so we screened active strains by plate streaking.

We used the following method to verify the expression results.

-Pick the bacterial plaque.

-Combined with water and glycerol, preserve.

-Inoculate the bacteria.

-Induce and culture for 4 hours, and measure fluorescence intensity by sampling.

-Continue culturing for 4 hours and measure fluorescence intensity.

Fig. 19. Fluorescence intensity of Tsinghua's expression result

8.10-9.6

We tested the ability of E. coli BL21 to be located on silicon column. We treated the columm with Tris-HCl buffer and filled it with induced E. coli BL21 and those hadn’t been induced respectively. Every hour we measured OD₆₀₀ of the solution for at least 6 hours to test the adsorbtion rate of the bacteria. We tried different conditions including the size and shape of the quartz sand and the size of the column.

The experiment experienced two stages as follows.

8.10-8.20

In the beginning we experimented with fine sand, however the interspace in fine sand is too small to fill sufficient bacterial solution. We tried adding more bacterial solution but it was proved to be difficult to obtain accurate data.

8.21-9.2

Then we replaced fine sand with coarse sand. We found that the effect of coarse sand adsorbtion depends on the roughness of sand surface. After testing several kinds of material, we got some good data. We stilled expected better results from fine sand experiments, but due to limited time we hadn’t got better data yet.

9.2-9.6

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. 20. 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.

8.26-9.20

We measured the adsorption effect of our adsorption system on rare earth element ions by ICP-OES. We had taken field samples of wastewater from the rare earth industry in our human practice investigation, and the target ions which were not found in drainage samples were prepared under laboratory conditions and measured together.

We used the following method to construct the system of adsorbtion system.

-Centrifugation of bacteria solution with 6000 rpm, then washing with MES solution twice and dissolving with MES buffer.

-Incubation for 2 hours, centrifugation and collect supernatant.

-Adding nitric acid to get rid of organic matter.

We conducted adsorption experiments on drainage with actual pH=3.9 and pH=6.9 respectively in order to confirm a proper condition for our system to work.

The specific schedule is as follows.

8.20-8.25

We conducted pre-experiment to determine the content of rare earth ions in the samples. Most of the rare earth element ions were included in samples.

8.26-8.31

We conducted pre-experiment and used Tb³⁺ to test whether our functional protein could absorb rare earth element ions.

9.1-9.15

Under laboratory conditions we tested the adsorption efficiency of different rare earth ions. We configurated rare earth ions solution with 150 µM, testing respectively and preliminarily obtaining the ability of adsorbtion.

Fig. 21. The Tb3+ adsorbtion of PmrL, PmrFP, GolSL, GolSFP

Fig. 22. The efficiency of PmrL absorbing La,Ce,Sm,Nd,Eu,Y,Gd,Sc

9.16-9.30

We measured the adsorption effect of our adsorbtion system on field samples of wastewater by ICP-OES. We tested with actual pH=3.9 and pH=6.9 respectively and confirmed an ideal condition for our system to work.

Fig. 23. Different REEs adsorbtion of PmrL system in industrial wastewater rich in REEs with different pH