As described in Design, our loop consists of a core “transport-chelation-redox reaction” system and an auxiliary “toxin-antitoxin” suicide system. Our project designed experiments to verify the “transport-chelation-redox reaction” system and verify the “suicide system” through modeling and other methods. The results are as follows:
1. Experiments in Synechocystis sp.PCC6803
The ultimate goal of our project is to use the modified Synechocystis sp.PCC6803 for the treatment of cadmium pollution in water bodies, therefore we carried out numerous experiments in Synechocystis sp.PCC6803 and obtained the following results:
1.1 Exploring the efficiency of cadmium uptake at different Synechocystis sp.PCC6803 concentrations
To preliminary investigate the cadmium uptake by wild Synechocystis sp.PCC6803, we designed different algae concentrations with OD values of 0.3, 0.6, 0.8, 1.1 and 1.4, respectively. We incubated the algae in low cadmium (0.1mg/L) and high cadmium (2mg/L). We measured the efficiency of cadmium absorption and growth. The results showed that (1) in the low cadmium group, the algae grew well, but in the high cadmium group, except for the algae with an OD of 1.4, the rest were inhibited, and the lower the concentration of algae, the more obvious the inhibition (Figure 1); (2) in the low cadmium group, the algae had a better effect of cadmium removal, while in the high cadmium group, except for the Synechocystis sp.PCC6803, with an OD value of 1.4, the effect of cadmium removal was not significant in other groups. What’s more, under high cadmium conditions, the cadmium uptake curve of Synechocystis sp.PCC6803 increases first and then decreases, indicating that cadmium is released after Synechocystis sp.PCC6803 death. (Figure 2). The above results show that wild Synechocystis sp. PCC6803 has a certain capacity to absorb cadmium, which is enhanced by the increase of initial algal concentration but is easily inhibited by high cadmium.
Figure 1: Growth at different algal concentrations (0.1mg/L, left) and (2mg/L, right).
Figure 2: Removal of cadmium at different algal concentrations (0.1mg/L, left) and (2mg/L, right).
1.2 Further investigation of cadmium resistance and uptake by Synechocystis sp.PCC6803
To further investigate the ability of wild Synechocystis sp.PCC6803 to remove and tolerate cadmium, we designed a cadmium concentration gradient of 0.1 mg/L, 0.5 mg/L, 1 mg/L, 2 mg/L, 5 mg/L and 10 mg/L. The absorbance at 730 nm was measured at eight time points (0.5h, 1h, 1.5h, 2h, 4h, 12h, 24h and 48h, respectively) to monitor the growth of Synechocystis sp.PCC6803. (figure3), it was observed that as the concentration of cadmium increased, the growth of Synechocystis sp.PCC6803 was inhibited more and more obvious. The OD value of algae in concentrations greater than or equal to 2 was lower than 0.7, which indicated that the growth of Synechocystis sp.PCC6803 was greatly inhibited. Afterward, the cadmium absorption efficiency of Synechocystis sp.PCC6803 under different cadmium concentrations were measured. The results showed that algae absorbed most of the cadmium in 0.5h and then entered the platform phase. And the cadmium absorption efficiency was lower under high cadmium condition than low cadmium condition, with the highest absorption efficiency at 0.1mg/L (figure4). The above results show that wild Synechocystis sp. PCC6803 can remove and absorb cadmium, but it needs further improvement.
Figure3: Growth of Synechocystis sp.PCC6803 at different cadmium concentrations
The growth of algae is inhibited when the concentration is greater than 2 mg/L, and algae’s growth is better in the case of low cadmium.
Figure 4: Cadmium uptake efficiency of Synechocystis sp.PCC6803 at different cadmium concentrations.
2. Embedding of Calcium-Alginate Microbeads (Ca-Alg MBs）
In order to improve the cadmium removal capacity and cadmium tolerance of Synechocystis sp.PCC6803, we proposed the Ca-Alg MBs embedding scheme, and the following are the experimental results we obtained:
2.1 Production of Ca-Alg MBs
Cell immobilization can effectively limit cell movement, reduce the escape of engineered algae, and prevent genetically engineered algae from harming the natural environment. In our project, we used sodium Ca-Alg MBs to embed the algae. We determined that the optimum concentration of sodium alginate is 3% by setting up a concentration gradient. The optimum concentration of calcium chloride is 4%. To ensure Ca-Alg MBs are square, we use pasteurization pipe and dropper to make our own rubber ball manufacturing pipe (Figure5). To facilitate modeling, ImageJ was used to calculate the diameter of Ca-Alg MBs (Table 1).
Figure 5: Cadmium uptake efficiency of Synechocystis sp.PCC6803 at different cadmium concentrations.
2.2 Exploring the role of the Ca-Alg MBs
At the same time, in order to verify the effect of Ca-Alg MBs embedding on the cadmium absorption efficiency of algae, we designed a comparative experiment between embedded algae, sodium Ca-Alg MBs and unembedded algae, finding that Ca-Alg MBs can improve the cadmium absorption efficiency of algae and the Ca-Alg MBs has a natural ability to absorb cadmium (Figure6A/B). At the same time, we found that the Ca-Alg MBs have a certain protective effect on algae in a high cadmium environment (Figure7). We speculate that the reasons are as follows: Ca-Alg MBs have three procedures called liquid phase transfer, Solid phase transfer, metathesis to absorb Cd and perform an excellent cadmium removal ability. So, it can immobilize our biological chassis and improve the cadmium elimination in high Cd by acting as a ‘buffer zone’.
Figure6: Effect of Ca-Alg MBs on the ability of algae to absorb cadmium.
We determined the cadmium uptake efficiency of Ca-AIg MBs, PCC6803, Ca-AIg MBs + PCC6803 under high and low cadmium conditions, respectively. The results showed that Ca-AIg MBs have some cadmium removal capacity and can protect PCC6803 under high cadmium conditions and improve its cadmium removal capacity (0.1mg/L, left) and (2mg/L, right).
Figure7: Protective effect of Ca-AIg MBs on algal growth in high cadmium environments
(0.1mg/L, left) and (2mg/L, right).
3.1.3 Life cycle of Ca-Alg MBs
Figure 8: During 36 hours, these MBs will bubbl with time or even burst due to the oxygen, suggesting that we need to recycle these MB in time to avoid the leak of PCC6803.
3. Experiments in E. coli
To further improve the cadmium uptake and resistance of Synechocystis sp.PCC6803, we designed the core “transport-chelation-redox reaction” loop. However, due to the difficulty of conducting an experiment in Synechocystis sp.PCC6803 compared with E. coli, we first verify our loop in E. coli
3.1 Successful construction of plasmids
Figure9: the backbone of our plasmid and the OptimumGeneTM algorithm we used to accomplish codon optimization
Figure10: our plasmids with different resistance
In our project, we selected nine proteins including three translocators: OsNRAMPS/MntH/IRT1, three chelating proteins: SmtA/hMT-1A/TMCd1 and three redox proteins: APX2/LOX1/Cu-SOD, and we successfully constructed the nine plasmids we needed to screen by using the vector pVZ322 (figure9), and we also added different resistances to our plasmids for next experiments (figure10).
3.2 Successful expression of our components in E. coli
We transfected E. coli using our constructed plasmids. We examined protein expression by western blot, and the results are as follows (figure11):
Figure11: the Western blotting results indicating the successful expression of our plasmids in E.Coli
3.3 Characteristics of the Engineering Synechocystis sp.PCC6803
To investigate the effect of successfully transferred plasmids on Synechocystis sp.PCC6803, we determined the growth curves of engineered and wild algae at certain cadmium concentrations, with the following results (figure12/13):
Figure12: Growth curves of chelated protein-expressed or redox protein-expressed or transport-chelation-redox protein-expressed E. coli in 0.1 mg/L of cadmium solution
We incubated E. coli expressing the protein TMCD1, hMT-1A, SmtA or APX2 and the E. coli expresses transport-chelation-redox protein in a cadmium solution concentration of 0.1 mg/L and determine the OD value in response to algal concentration. The results showed that these proteins could improve algae cadmium tolerance and protect their growth in a high cadmium environment.
To further verify our proteins' role, we incubated E. coli expressing hMT-1A, MntH, MntH&TMCd1&APX2 in different cadmium concentrations. The results showed that E. coli expressing transport-chelating-antioxidant and chelating proteins had higher cadmium tolerance, while E. coli expressing only transport proteins had poorer cadmium tolerance due to more cadmium transfer (figure5).
Figure 13: Growth curves of modified E. coli at different cadmium concentrations.
3.4 Efficiency of cadmium uptake in modified E. coli
To verify the change in cadmium uptake efficiency of E. coli after successful genetic modification, We cultured E. coli successfully expressing hMT-1A, MntH, MntH&TMCd1&APX2 in a medium with a cadmium concentration of 0.1 mg/L, determined the cadmium concentration at different times and plotted the cadmium uptake curve (figure14). The results showed that the expression of the transport protein MntH increases the efficiency of cadmium uptake by E. coli but substantially decreases its cadmium tolerance. And the E. coli was successfully expressing MntH&TMCd1&APX2 absorbed more cadmium than those expressing MntH alone and empty plasmid.
Figure 14: Cadmium uptake curve of engineered E. coli.
4. Enhancing safety
To further reduce the environmental damage caused by genetically modified algae, we designed a toxin-antitoxin suicide system. Blue light inhibition element was used, which can down-regulate the expression of the downstream protein. Due to the covid-19, the verification experiment of this system could not be carried out in time. Still, we have proved the system from the perspective of mathematical modeling. The results showed that the system can inhibit the toxin expression under blue light, and the algae can grow naturally. However, When the blue light disappears, the toxin is expressed in large quantities, leading to the algae’s death.
To further improve our project, the following supplement and efforts will be carried out in nearly future:
4.1 Further verify the optical control system in the laboratory
In our experimental part, although we have confirmed the effect of the visual control system with the modeling method, its real impact remains to be further verified by experiments. In the future, we will synthesize optical control elements and demonstrate them through the transformation, selection and amplification and other molecular biology standard processes.
4.2 Explore the growth curve of the modified algae
Although the modified alga can remove cadmium effectively, its growth curve under different cadmium concentrations is unknown. In the following experiments, we will design different cadmium concentration gradients to observe the growth of algae to provide data support for the use cycle of subsequent engineering algae used in the environment
4.3 Verify safety and practicality in the real environment
Although our engineered algae’s cadmium removal efficiency has been verified in natural water bodies, its actual performance in large-scale water bodies is unknown. Next, we will select the bodies of water polluted by cadmium to ascertain the practicality of our algae
After completing the above experiment supplement, we will promote our algae in various ways and apply it in the real environment
In subsequent experiments, we will use a molecular evolutionary approach to screen our pre-selected proteins. Express the cadmium ion probe in Synechocystis sp.PCC6803, point mutates the domains related to cadmium ion recognition and express them in the algae. And then, screen the proteins with high fluorescence intensity of the probe by flow cytometry, isolate and sequence them to determine and select the more efficient proteins.
5. Considerations for replicating our experiments
The experiments we conducted are all routine experiments in molecular biology with standard operating procedures. However, the following two points should be noted during the replication of our experiments:
5.1 The protection of toxic substances
In our experiments, a large number of poisonous substances are used, including the toxin RelB…. Please pay attention to the protection
5.2 The treatment of engineering algae
After the modification, the engineered Synechocystis sp.PCC6803 has a robust antioxidant ability, and its survival ability has been enhanced, too. Therefore, when dealing with it, be careful not to let them escape and cause environmental damage.
The core of our project is to improve the cadmium removal efficiency of Synechocystis sp.PCC6803 through synthetic biology methods under the premise of ensuring biosafety. Based on the above experimental results, we can draw the following conclusions:
1. Synechocystis sp. PCC6803 has a particular ability to remove cadmium. The efficiency of cadmium removal increases with an increasing initial concentration of Synechocystis sp. PCC6803.
2. Synechocystis sp. PCC6803 grows well under low cadmium conditions but is inhibited at cadmium concentrations above 2 mg/L.
3. Ca-AIg MBs have a particular ability to remove cadmium and can improve the ability of Synechocystis sp. PCC6803 to resist and remove cadmium.
4. The components we designed could be expressed in prokaryotes and can significantly improve cadmium resistance and removal.
5. Mathematical modeling demonstrates that our light-controlled suicide system could ensure the biosafety of our modified organisms
The above experimental results show that our design could help prokaryote effectively remove cadmium from the water with good biological safety