Part:BBa_K5205013

J23100-Non-OmpA-OsMTI-1b

This is a complete expression cassette consisting of a strong constitutive promoter BBa_J23100, a fusion rice metallothionein OsMT1 BBa_K5205004, 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
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

The fusion of Non-OmpA with OsMTI-1b enables the metallothionein to be displayed on the E. coli cell surface BBa_K5205004, allowing the fusion OsMT1 to effectively bind heavy metal ions in the environment. We expressed this fusion protein in E. coli BL21 to turn it into 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%. In OsMT1, the cadmium binding capacity increased significantly in higher concentrations. At 0.001 mM, it exhibited the highest removal rate of 88.53% and maintained a high removal rate of 74.60% 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.

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%. In OsMT1, removal rates exceeded 95% at 0.001 and 0.01 mM, but saturation occurred at 0.1 mM.

To conclude, the expression of fusion OsMT1 dramatically increased the ability of heavy metal removal by E. coli DH5α.