Our project focused on removing heavy metal ions from water by engineering E. coli to express rice metallothionein (OsMTI-1b) for metal binding and a urease gene cluster from Sporosarcina pasteurii for Microbiologically Induced Calcite Precipitation (MICP). In tests, both OsMT1 and the urease gene cluster functioned effectively in our engineered bacteria, significantly increasing the removal rates of Cd²⁺ and Pb²⁺. The strain expressing both genes demonstrated the best overall performance, with OsMT1 excelling at lower concentrations and the urease gene cluster being more efficient at higher concentrations, achieving maximum removal rates of 85.78% for cadmium and 98.98% for lead.
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Type |
Description |
Length |
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|
Basic |
OsMTI-1b |
219 bp |
|
|
Basic |
Non |
27 bp |
|
|
Basic |
Partial OmpA |
342 bp |
|
|
Composite |
Non-OmpA |
369 bp |
|
|
Composite |
Non-OmpA-OsMTI-1b |
588 bp |
|
|
Basic |
ureA |
303 bp |
|
|
Basic |
ureB |
381 bp |
|
|
Basic |
ureC |
1713 bp |
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|
Basic |
ureE |
444 bp |
|
|
Basic |
ureF |
663 bp |
|
|
Basic |
ureG |
636 bp |
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|
Basic |
ureD |
828 bp |
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|
Composite |
Urease gene cluster |
5326 bp |
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|
Composite |
J23100-Non-OmpA-OsMTI-1b |
671 bp |
|
|
Composite |
J23100-Urease gene cluster |
5409 bp |
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|
Composite |
J23100-Fusion OsMTI-1b-Urease gene cluster |
5997 bp |
Best Composite Part
BBa_J23100 Promoter-Fusion OsMT1-Urease gene cluster-T7 Terminator
This is a complete expression cassette consisting of a strong constitutive promoter (BBa_J23100), a fusion rice metallothionein OsMT1 (BBa_K5205004), a urease gene cluster from Sporosarcina pasteurii DSM33 (BBa_K5205012), and a T7 terminator (BBa_K731721). The fusion of Non-OmpA with OsMTI-1b enables the metallothionein (metal-binding protein) to be displayed on the E. coli cell surface, allowing the fusion OsMT1 to effectively bind heavy metal ions in the environment. The urease gene cluster encodes for the urease enzyme complex, which is crucial for catalyzing the hydrolysis of urea into ammonia and carbon dioxide, a key step in the process of microbially induced calcite precipitation (MICP). MICP can precipitate heavy metals like cadmium and remove them from the water. By introducing the fusion OsMT1 and the urease gene cluster into E. coli, E. coli can be engineered to be a heavy metal remover.
Characterization
Hg removal
We prepared LB media containing mercury(II) nitrate concentrations ranging from 0 to 10 mM in 10-fold dilutions. A 1% inoculum of the engineered E. coli was subcultured into each mercury-containing medium and incubated overnight at 37 °C for 24 hours. On day 2, OD600 measurements were taken for each sample (Figure 1A). The results showed no increase in mercury tolerance (at least not greater than 10-fold) in the engineered strain compared to the original DH5α.
Figure 1. A. Growth of E. coli DH5α-based strains in liquid LB containing Hg²⁺; B. Removal rates of Hg²⁺ by E. coli in 24 hours. “N/A” stands for “not applicable,” as no data was collected from 0.001 to 10 mM due to the absence of cell growth.
The supernatants from the 24-hour cultures were collected and sent to Convinced-test Tech. Co., Ltd (Nanjing, Jiangsu, China) for Hg²⁺ concentration analysis. Mercury removal rates were calculated and are shown in Figure 1B. The results indicated that at a very low concentration of 0.0001 mM, all strains demonstrated a similar mercury removal rate of about 87%, suggesting that the expression of OsMT1 and the urease gene cluster did not enhance mercury removal at the concentration the bacteria could tolerate.
Cd Removal
Cadmium removal tests were conducted following the same protocol as for mercury. The results from Figure 2A showed no increase in cadmium tolerance (at least not greater than 10-fold) in the engineered strain compared to the original DH5α.
Figure 2. A. Growth of E. coli DH5α-based strains in liquid LB containing Cd²⁺; B. Removal rates of Cd²⁺ by E. coli in 24 hours. “N/A” stands for “not applicable,” as no data was collected from 1 to 10 mM due to the absence of cell growth.
The results indicated that at a very low concentration of 0.0001 mM cadmium, all strains demonstrated a similar removal rate of approximately 80%. In OsMT1, the cadmium binding capacity increased significantly. At 0.001 mM, it exhibited the highest removal rate and maintained a high removal rate at 0.1 mM. However, the binding capacity of the OsMT1 protein on the cell surfaces was eventually saturated at 0.1 mM. In summary, OsMT1 was most effective at cadmium concentrations below 0.01 mM. For Ure, there was no noticeable change in removal rate compared to DH5α at 0.001 mM, likely due to the low efficiency of the chemical reaction (formation of cadmium carbonate) when the concentration of one reactant is very low. The removal rate increased as the cadmium concentration rose from 0.01 to 0.1 mM. In conclusion, urease is more suitable for high-concentration conditions and was most effective when cadmium concentrations exceeded 0.1 mM. For OsMT1-Ure, it appears that both mechanisms acted independently, as the removal rates were close to the sum of the individual performances of OsMT1 and Ure.
Pb Removal
Lead removal tests were conducted following the same protocol as for mercury and cadmium. The results from Figure 3A showed no increase in lead tolerance (at least not greater than 10-fold) in the engineered strain compared to the original DH5α.
Figure 3. A. Growth of E. coli DH5α-based strains in liquid LB containing Pb²⁺; B. Removal rates of Pb²⁺ by E. coli in 24 hours. “N/A” stands for “not applicable,” as no data was collected on 10 mM due to the absence of cell growth.
The Pb²⁺ removal rates were similar to Cd²⁺. At a low concentration of 0.0001 mM, all strains showed a similar removal rate of around 90%, driven by the intrinsic metal-binding/absorbing systems. In OsMT1, removal rates exceeded 95% at 0.001 and 0.01 mM, but saturation occurred at 0.1 mM. Ure exhibited no significant difference from DH5α at low concentrations but showed a high increase in removal rate at higher concentrations (0.1 to 1 mM). OsMT1-Ure showed the best overall performance, with OsMT1 handling lower concentrations and Ure managing higher concentrations, making the combination more effective across a wider range of lead concentrations.
Removal Rate
Based on previous tests, we found that OsMT1-Ure was the most effective strain for heavy metal removal. In this experiment, we tested the removal rate by time, by adding Cd²⁺ and Pb²⁺ to overnight cultures of E. coli DH5α expressing OsMT1-Ure. Samples were taken every 2 hours to monitor the changes in the removal rate.
Figure 4. Changes in the removal rate of Cd²⁺ and Pb²⁺ by E. coli DH5α expressing OsMT1-Ure.
Results showed that at a low concentration of 0.01 mM, the removal of both lead and cadmium was completed in less than 2 hours. At this concentration, the removal was primarily due to OsMT1 binding to the heavy metal ions directly, which explains the rapid reaction. At a higher concentration of 0.1 mM, where the urease gene cluster was
dominating, the removal took about 4 hours due to the time required for urea hydrolysis.
In conclusion, OsMT1-Ure demonstrated the best overall performance in heavy metal removal, proving efficient across a broader range of concentration levels, with maximum removal rates of 85.78% for cadmium and 98.98% for lead.
In the future, the iGEM community can expand our composite part by incorporating more genes.