Introduction
We want to absorb the residual REEs in the waste water of the rare earth industry and be able to recover them. To this end , we designed several components and four system pathways to achieve this goal. In the construction stage, we used homologous recombination to construct the pathway. Although we encountered some problems, we finally achieved certain results through continuous attempts and summaries, which also provided some experience for the subsequent teams.
Cycle1: Construct by the homologous recombination method
1.1 Design
In order to construct a well-functioning systemic pathway, we hope that a single plasmid can be used to complete the two tasks of sensing rare earth ions and enriching rare earth ions, and make the engineered bacteria more sensitive to the sensing of rare earth elements in the environment so that the enrichment could be more efficient. We use existing components with independent functions to connect multiple components into systemic pathways with complete functions to construct complete plasmids to achieve our goal.
We set the stage for building the entire systemic pathway when we built the individual components. When we designed it, we wanted to be able to use homologous recombination to build our fully functional plasmids. Firstly in the PmrL and PmrFP pathways , we added the first homologous arm to the downstream of the linearized fragment containing pmrA gene and kanamycin resistance gene as vector and upstream of pmrB(LanM) and pmrB(LBT-LanM). Then a second homologous arm was added to the downstream of pmrB(LanM), pmrB(LBT-LanM) and the upstream of pmrC promoter, after which a third homologous arm was added to the downstream of PmrC and upstream of oprf-sitag-LanM and oprf-sitag- dLBT-LanM .
In the GolSL and GolSFP pathways, we added the first homologous arm to the downstream of the linearized fragment containing kanamycin resistance gene and upstream of golS-PgolB. A second homologous arm was added to the downstream of golS-PgolB and upstream of oprf-sitag-LanM and oprf-sitag-dLBT-LanM. Likely, we added a third homologous arm to the downstream of oprf-sitag-LanM and oprf-sitag-dLBT-LanM and upstream of the linearized vector T7-KanR. These homology arms are constructed using PCR using different primers to add to different gene segments. These homologous arms with specificity ensure the feasibility of the experiment in principle.
Fig. 1. The diagram of constructing the plasmid inserted with PmrL by multi-fragment homologous recombination
Fig. 2. The diagram of constructing the plasmid inserted with GolSL by multi-fragment homologous recombination
1.2 Build
Using the most efficient seamless cloning principle at present, we selected homologous recombinase to construct the pathway, designed primers in strict accordance with the instructions, and carried out the operation process to construct our plasmid. We used PCR to obtain gene fragments from plasmids containing elements, and then verified and purified by gel recovery. Taking the construction of PmrL system pathway as an example, we first used PCR to obtain the linearized fragment pmrA-KanR as vector and three gene fragments pmrB, PmrC, oprf-sitag-LanM. The purified vector and each gene fragment were verified by re-gel recovery. At last, the vector and fragments were connected by homologous recombinase, transformed and cultured. Finally we validated the colony of bacteria to obtain our plasmid.
We attempted to ligate multiple fragments directly to the vector into complete plasmids using homologous recombinases.Take the GolSL system pathway as an example: Firstly, we used PCR to obtain the linearized vector T7-KanR and two gene fragments—golS-PgolB and oprf-sitag-LanM. Then gel recovery was used to verify the purification of the vector and gene fragments. Finally, homologous recombinase was used to directly link the linearized vector T7-KANR with two fragments of golS-PgolB and oprf-sitag-LanM to construct a complete systemic pathway. Then, we transferred the homologous recombination products into TOP10F’ and inoculated onto LB plates containing 50 µg/mL Kana for screening. When the single colony grew, we picked up the colony for PCR verification. After the target band was observed, the bacterial solution was mixed with a 1:1 mixture of glycerol and water, and the seed was preserved to -80℃.
1.3 Result
We tried to construct our system pathway by multi-fragment homologous recombination. However, after several attempts, the effect was not ideal and the success rate was low. Only plasmids of GolSL system pathway were successfully constructed: T7-golS-T7 Terminator-PgolB-oprf-sitag-LanM-T7 Terminator. However, PmrL, PmrFP and GolSFP pathways were not successfully constructed.
Fig. 3. The result of PCR identification of GolSL plasmid shows that there is bright band at about 4000 bp, and the length of GolSL plasmid is 4200 bp, which proves that we have successfully constructed the plasmid of GolSL system pathway.
1.4 Learn
After several attempts, we found that the success rate of direct multi fragment homologous recombination was not high. But fortunately, we have achieved some results and constructed one of the four targeted pathways, GolSL . We carefully analyzed and summarized the reasons for the failure. We believe that the success rate of direct homologous recombination is not high due to the large number of gene fragments in PmrL, PmrFP and GolSFP pathways. We think that overlap could be attempted to solve this problem.
Cycle2: Construct by overlap and homologous recombination method
2.1 Design
After many failed attempts, we decided to use overlap to connect multiple short fragments together and insert them into the carrier after purification, so as to avoid the problem of low success rate of homologous recombination of multiple fragments. In the initial design, we have added corresponding homologous arms to each fragment to ensure the feasibility of our experiment in principle. Meanwhile, we have obtained the purified fragments of the corresponding parts in the previous experiments. For PmrL and PmrFP: since pmrB(LanM) or pmrB(LBT-LanM) and PmrC have the same first homologous arm ,we can overlap to connect them. PmrC has the same second homologous arm with oprf-sitag-LanM and oprf-sitag- dLBT-LanM. oprf-sitag-LanM or oprf-sitag- dLBT-LanM can be ligated to previously ligated pmrB（LanM）-PmrC or pmrB(LBT-LanM)-PmrC for single fragment homologous recombination. For GolSFP system: the downstream of golS-PgolB and the upstream of oprf-sitag- dLBT-LanM were added the same homologous arm, which can be connected by overlap. Thus, single fragment homologous recombination can be carried out to improve the success rate.
Fig. 4. PmrL fragment overlay diagram. First, connect 1 (pmrB), 2 (PmrC) by overlap to get 4 (pmrB-PmrC), and then connect 4 (pmrB-PmrC) with 3 (oprf-sitag-LanM) by overlap to connect 1,2,3 fragments.
2.2 Build
Due to the large number of gene fragments in PmrL and PmrFP pathways, the success rate of direct homologous recombination was low. Therefore, each gene fragment was first connected by overlapping extension, and finally the connected fragment was homologous recombination with the vector to obtain the plasmid. Taking the construction of PmrL system pathway as an example, firstly linear fragments containing pmrA gene and kanamycin resistance gene as vectors and three gene fragments pmrB, PmrC and oprf-sitag-LanM were obtained by PCR, and then the vector and each gene fragment were verified and purified by gel recovery. Due to the large number of fragments, we used overlap to connect pmrB and PmrC first, and then pmrB-PmrC and oprf-sitag-LanM. Finally, we used homologous recombinase to connect the linearized vector containing pmrA gene with the connected fragment to construct the system pathway. Then, the homologous recombination products were transferred into TOP10F 'and inoculated onto LB plates containing 50µg/mL Kana for screening. When the single colony grew, we picked up the colony for PCR verification. After the target band was observed, the bacterial solution was mixed with a 1:1 mixture of glycerol and water, and the seed was preserved to -80℃.
2.3 Result
After several attempts, we successfully constructed plasmids of PmrL , PmrFP and GolSFP systematic pathways: 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- dLBT-LanM - T7 Terminator.The three plasmids were constructed by overlap and then homologous recombining of a single fragment.
Fig. 5. The left figure is the PCR identification result of PmrL plasmid shows that there is bright band at about 4500 bp, and the length of PmrL plasmid is 4289 bp, which proves that we have successfully constructed the plasmid of PmrL system pathway
The middle figure is the PCR identification results of PmrFP plasmid shows that there is bright band at about 4500 bp, and the length of PmrFP plasmid is 4439 bp, which proves that we have successfully constructed the plasmid of PmrFP system pathway.
The right figure is the result of PCR identification of GolSFP plasmid shows that there is bright band at about 4500 bp, and the length of GolSFP plasmid is 4350 bp, which proves that we have successfully constructed the plasmid of GolSFP system pathway.
2.4 Learn
We use overlap to connect multiple short fragments and then carry out homologous recombination of single segments to successfully construct PmrL, PmrFP and GolSFP system pathways so that all the four pathways of our target are successfully constructed. However, we also found some problems in the process of constructing plasmids. For example, the plasmid T7-pmrA- T7-pmrB(FP)-T7 Terminator-PmrC-oprf-sitag-FP-T7 Terminator constructed by homologous recombination would have multiple bands. However, the presence of the correct band also indicated that we had successfully constructed the plasmid. In the subsequent exploration and discussion, we realized that the upstream primers we designed for sequencing were bound to the His-Tag before pmrA, but we also added His-Tag before the C terminus of Oprf-Sitag- dLBT-LanM in the designed systemic pathway, and the primers could also bind to it. This created the multi-band situation. After realizing this problem, we redesigned our primers and selected a more specific fragment design primer. After changing the primer, we successfully solved the problem of multiple bands.
The recovery of rare earth elements is of great significance for the protection of the Earth environment and the full utilization of resources, and we are encouraged to make some contributions in this respect by using synthetic biology. We will continue conducting this subject and are willing to do our part for the development of synthetic biology.
Expression validation
3.1 Sensing device
3.1.1 Design
In order to enable our engineered bacteria to directly sense the rare earth elements in wastewater, we inserted LanM, a lanthanide sensing protein, or LanM along with dLBT, which can capture the rare earth elements, into the membrane protein PmrB in the Pmr system. In addition, we also added a His-tag to the end of the pmrA sequence for later protein purification and identification. At the same time, in order to enhance our expression effect, we selected the potent promoter T7 to initiate the expression of proteins PmrA and PmrB, so as to enhance the expression efficiency of the Pmr two-component system.
3.1.2 Built
We transferred the constructed plasmid into E. coli BL21 and inoculated onto LB plates containing 50µg/mL Kana for screening. When the single colony grew, we picked up the colony for PCR verification. After the target band was observed, we mixed the bacterial solution with a 1:1 mixture of glycerol and water, and the seed was preserved to -80℃ and verified the expression.
3.1.3 Test
We verified that our plasmid was successfully transferred to BL21 by colony PCR. The specific method is to culture the selected single colony of E. coli in a shake flask containing 1ml liquid LB with Kana, and conduct PCR verification after the bacteria is amplified for avoiding PCR failure caused by the engineering bacterium concentration is too low, or PCR success but strain amplification failure cases.
Fig. 6. The PCR of PmrL, PmrFP, GolSL and GolSFP colonies all had bright and correct bands, which proved that the transformation was successful
After colony PCR identified bright and correct bands, we preserved them for expanded culture and induction of expression. Firstly, we expanded the number of bacteria in the liquid LB medium containing Kana. When the OD600 of medium reached 0.6-0.8, we added IPTG and Tb3+ into PmrL and PmrFP system for induction and expression, and then cultured them for 13h. After centrifugation, the cells were treated with PBS buffer and broken by ultrasound. After 1h of low-temperature centrifugation at 12000 rpm, we added TDSET buffer to obtain membrane protein solution , and then SDS-PAGE was performed to test. Finally, we successfully detected the target band of membrane protein PmrB at 38kDa.
Fig. 7. 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 to show PmrB successful expression
In addition to SDS-PAGE verification of membrane proteins, we also tried to purify and verify PmrA proteins in cells. After the engineering bacteria were expanded and induced to express, we centrifuged the samples and broke cells. Then we extracted and purified proteins by nickel column, using SDS-PAGE to determine whether there were target products. Finally, we successfully observed the target protein band on the gel at 26.8kDa. But the color was light and the concentration was low.
Fig. 8. After purifying of PmrL by nickel column, SDS-PAGE verification showed that there was a band at 25-30kDa that was significantly different from that of throughflow and impurity washing, which confirmed that PmrA was expressed in cells
For the GolS system, after obtaining the bright and correct bands identified by colony PCR, we preserved them, cultured them and induced the expression. Firstly, we expanded the number of bacteria in liquid LB containing Kana. When the OD600 reached 0.6-0.8, IPTG and Cu2+ were added to GolSL and GolSFP systems for induction, and then cultured them for 13h. We centrifuged the samples and broke cells. Then we extracted and purified proteins by nickel column, using SDS-PAGE to determine whether there were target products. Finally, we detected that the target band was observed at 17.8kDa where GolS was located.
Fig. 9. After purifying of 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
3.1.4 Learn
After SDS-PAGE, we successfully observed the target band of PmrB protein at 38kDa, but the band of PmrA was light in color and low in concentration. After analysis, we believe that PmrA may be phosphorylated by PmrB in the cell then activating PmrC, which caused PmrA to be decomposed and recycled by the cell, leading to the low concentration of PmrA in the cell. However, due to the characteristics of Pmr system, we can further prove the expression of PmrA by the expression of capture part.
3.2 Capture device
3.2.1 Design
In order to enable our engineering bacteria to directly capture lanthanide elements in wastewater, we used Oprf, an anchor protein, to fix our composite protein on the outer side of the cell membrane. At the same time, to facilitate our subsequent recovery of REEs, we added Si-tag after Oprf to enable it to absorb on the silica column. Finally, LanM or the fusion protein LanM and dLBT are responsible for the adsorption of lanthanide elements. In addition, we also added a His-tag to the C-terminus of Oprf to facilitate the identification of whether the protein was successfully anchored on the cell surface.
3.2.2 Built
We transferred the constructed plasmids into E. coli BL21 and inoculated it onto LB plates containing 50µg/mL Kana for screening. When the single colony grew, we picked up the colony for PCR verification. After the target band was observed, we mixed the bacterial solution with a 1:1 mixture of glycerol and water, and the seed was preserved to -80℃ and verified the expression.
3.2.3 Test
3.2.3.1 Validation of membrane protein expression
After colony PCR identifies bright and correct bands, we preserved the engineering bacteria for expanded culture and induction of expression. The engineered bacteria of the four pathways were firstly inoculated in LB liquid medium containing Kana for expansion culture. When the OD600 of medium reached 0.6-0.8, we added IPTG and rare earth ions into PmrL and PmrFP system for induction. We tried to add Tb, Sc, Ce, La and Yb to induce and we observed the expression. We added an appropriate amount of IPTG and Cu2+ to GolSL/GolSFP to induce the expression. After 13 hours of incubation, we took appropriate amount of bacterial solution to extract membrane protein, and used SDS-PAGE for verification. Finally, we successfully observed the correct band of target protein at 63kDa. At the same time, we found that the effects of expression of PmrL and PmrFP system were best when Tb3+ was used for induction, so Tb3+ was used for induction in subsequent experiments.
3.2.3.2 Verification of Si-tag adsorption
After determining the successful expression of our capture part, we tried to test the adsorption capacity of engineered bacteria using a column filled with silica, so as to verify whether our Si-tag could work properly and achieve our expected goal. After induction , we added the bacteria solution to the silica column balanced with Tris buffer and incubated it for 6h. During this period, we took 2ml of the effluent every 1h 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 adsorption.
Considering that the relative surface area of fine sand is large, we first tried to fill our chromatographic column with fine sand. After many attempts, we found that although the relative surface area of fine sand is large, more bacteria will be absorbed on the silica chromatographic column through physical adsorption. Even the control group that has not been induced to express has a high adsorption rate, and it is difficult to determine our real adsorption situation. In view of this situation, we replaced the coarse sand with a larger diameter for the adsorption experiment. After the fine sand was replaced with coarse sand, the experimental effect was significantly improved. The adsorption of the control group and the experimental group was significantly different, but the adsorption effect of the experimental group did not meet our expectations. After our discussion, we found that the roughness of the coarse sand surface may also affect the adsorption of engineering bacteria, so we used a variety of coarse sand with different roughness but almost the same diameter to fill our chromatographic column for adsorption experiments. After many attempts, gratifying results have been achieved, among which the adsorption rate of the experimental group using the roughest coarse sand reached 89%, meeting our expectations.
Fig. 12. OD value of PmrL absorbed by coarse sand. The red is the control group, and the black is PmrL. It can be seen that the OD value decreased significantly after 6 hours, which can prove that the adsorption capacity of Si tag is very high
3.2.3.3 Verification of adsorption capacity of rare earth elements
After confirming that all other parts of the engineered bacteria work effectively, we need to measure the ability of the fusion protein to absorb REEs. We treated the induced bacterial solution with MES buffer, so as to obtain the experimental system to absorb rare earth ions. Subsequently, we added an appropriate number of rare earth ions to the adsorption 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. We added Tb3+ firstly to test the REEs adsorption of the engineering bacteria of the four pathways. After several attempts, the engineered bacteria of our PmrL system pathway got an optimal outcome--the averaged Tb3+ adsorption efficiency was 84%.
Fig. 13. The Tb3+ adsorption of PmrL, PmrFP, GolSL, GolSFP
Due to time limitation, we chose the engineering bacteria of PmrL system pathway to conduct following experiments. After verifying the engineered bacteria's ability of absorbing REEs, we tried adding different kinds of REEs to test the adsorption rate of our target ions of different REEs.
Fig. 14. The efficiency of PmrL absorbing La,Ce,Sm,Nd,Eu,Y,Gd,Sc
After successfully verifying the adsorption capacity of our engineering bacteria in the buffer liquid system, we tried applying our engineering bacteria to practice. We used MES buffer solution to treat the bacterial solution that had been induced, and then added it to industrial wastewater rich in rare earth elements and adjusted pH，incubating at 100 rpm, 30℃ for 2h before centrifugation. We used ICP-OES to determine the concentration of rare earth ions and the average adsorption of Sm3+ was up to 93%.
Fig. 15. Different REEs adsorption of PmrL system in industrial wastewater rich in REEs with different pH
3.2.4 Learn
The SDS-PAGE test confirmed that there was a correct band of the capture protein at 63kDa. At the same time, the immunofluorescence labeling also enabled us to successfully confirm that the capture protein was in the correct position —outside the cell membrane. The engineering bacteria could also absorb on the silicon column and be continuously recycled. We also achieved the expected effect on the determination of the adsorption capacity of REEs. These all confirmed that the four system pathways we constructed were successfully expressed. Finally, we also put the engineering bacteria into practice and added them to the wastewater rich in rare earth ions, and achieved success. Through this series of experiments, we have successfully constructed an engineering E. coli that can not only efficiently absorb REEs, but also recover REEs. However, there are still some parts of our project that can be optimized. We believe that even after the iGEM competition, we will continue to promote our project and make efforts to use synthetic biology to improve the world environment and make rational use of resources.
