Part:BBa_K5205014

J23100-Urease gene cluster

This is a complete expression cassette consisting of a strong constitutive promoter BBa_J23100, a urease gene cluster from Sporosarcina pasteurii DSM33 BBa_K5205012, and a T7 terminator BBa_K731721.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 4654
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3438
  Illegal BamHI site found at 5155
  Illegal XhoI site found at 4192
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4450
  Illegal BsaI.rc site found at 496
  Illegal BsaI.rc site found at 2224
  Illegal BsaI.rc site found at 4739
  

Usage and Biology

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 (Qasem et al., 2021). By introducing the urease gene cluster from S. pasteurii into E. coli, E. coli can be engineered to be a heavy metal remover.

Characterization

2024 Hangzhou-SDG Team characterized this part with heavy metal removal

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 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 from Figure 2B indicated that at a very low concentration of 0.0001 mM cadmium, all strains demonstrated a similar removal rate of approximately 80%. 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, reaching 55.04%. In conclusion, urease is more suitable for high-concentration conditions and was most effective when cadmium concentrations exceeded 0.1 mM.

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%. 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), peaking at 96.25%.

To conclude, the expression of the urease gene cluster dramatically increased the ability of heavy metal removal by E. coli DH5α.

References

Qasem, N. A. A., Mohammed, R. H., & Lawal, D. U. (2021). Removal of heavy metal ions from wastewater: a comprehensive and critical review. npj Clean Water, 4(1), 36. https://doi.org/10.1038/s41545-021-00127-0