Difference between revisions of "Part:BBa K5205015"

 
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This is a complete expression cassette consisting of a strong constitutive promoter [https://parts.igem.org/Part:BBa_J23100 BBa_J23100], a fusion rice metallothionein OsMT1 [https://parts.igem.org/Part:BBa_K5205004 BBa_K5205004], a urease gene cluster from Sporosarcina pasteurii DSM33 [https://parts.igem.org/Part:BBa_K5205012 BBa_K5205012], and a T7 terminator [https://parts.igem.org/Part:BBa_K731721 BBa_K731721].  
 
This is a complete expression cassette consisting of a strong constitutive promoter [https://parts.igem.org/Part:BBa_J23100 BBa_J23100], a fusion rice metallothionein OsMT1 [https://parts.igem.org/Part:BBa_K5205004 BBa_K5205004], a urease gene cluster from Sporosarcina pasteurii DSM33 [https://parts.igem.org/Part:BBa_K5205012 BBa_K5205012], and a T7 terminator [https://parts.igem.org/Part:BBa_K731721 BBa_K731721].  
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'></span>
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===Sequence and Features===
 
<partinfo>BBa_K5205015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5205015 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K5205015 parameters</partinfo>
 
<partinfo>BBa_K5205015 parameters</partinfo>
 
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<!-- Add more about the biology of this part here-->
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===Usage and Biology===
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The fusion of Non-OmpA with OsMTI-1b enables the metallothionein to be displayed on the E. coli cell surface [https://parts.igem.org/Part:BBa_K5205004 BBa_K5205004], 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 (Qasem et al., 2021). By introducing the fusion OsMT1 and the urease gene cluster into E. coli, E. coli can be engineered to be a heavy metal remover.
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===Characterization===
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'''2024 Hangzhou-SDG Team characterized this part with heavy metal removal'''
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'''Hg removal'''
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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.
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<html>
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<body>
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<figure>
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<div class = "center">
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<center><img src = "https://static.igem.wiki/teams/5205/parts/15-1.png" style = "width:400px"></center>
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</div>
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<figcaption><center>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. </center></figcaption>
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</figure>
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</body>
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</html>
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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.
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'''Cd Removal'''
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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.
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<html>
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<body>
 +
<figure>
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<div class = "center">
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<center><img src = "https://static.igem.wiki/teams/5205/parts/15-2.png" style = "width:400px"></center>
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</div>
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<figcaption><center>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. </center></figcaption>
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</figure>
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</body>
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</html>
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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.
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'''Pb Removal'''
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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.
 +
 +
<html>
 +
<body>
 +
<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.wiki/teams/5205/parts/15-3.png" style = "width:400px"></center>
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</div>
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<figcaption><center>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.  </center></figcaption>
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</figure>
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</body>
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</html>
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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.
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'''Removal Rate'''
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Based on previous tests, we found that OsMT1-Ure was the most effective strain for heavy metal removal, efficiently managing both low and high concentrations. 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.
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<html>
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<body>
 +
<figure>
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<div class = "center">
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<center><img src = "https://static.igem.wiki/teams/5205/parts/15-4.png" style = "width:400px"></center>
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</div>
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<figcaption><center>Figure 4. Changes in the removal rate of Cd²⁺ and Pb²⁺ by E. coli DH5α expressing OsMT1-Ure.  </center></figcaption>
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</figure>
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</body>
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</html>
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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.
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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.
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===References===
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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

Revision as of 04:31, 24 September 2024


J23100-Fusion OsMTI-1b-Urease gene cluster

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.

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 5242
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4026
    Illegal BamHI site found at 5743
    Illegal XhoI site found at 4780
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5038
    Illegal BsaI.rc site found at 1084
    Illegal BsaI.rc site found at 2812
    Illegal BsaI.rc site found at 5327


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. 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 fusion OsMT1 and the urease gene cluster 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 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, efficiently managing both low and high concentrations. 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.


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