Week 1 (7.8-7.12)

1. Sporosarcina pasteurii DSM33 resuscitation and subculturing:

1) Prepare liquid LB media (Solarbio, Beijing, China);

2) Prepare urea (500 g/L in water) stock solution. Sterilize using 0.22 µm filters (BKMAM, Changde, Hunan, China);

3) Add urea to LB media to reach a 25 g/L final concentration.

4) Subculture S. pasteurii DSM33 from the stock culture in the LB-urea media;

5) Culture at 30 ℃, 200 rpm.

Notes & Results:

Succeeded. Media became cloudy after 24 hours.

 

2. PCR amplification of the urease gene cluster from S. pasteurii DSM33:

1) 50 μL PCR:

20 μL deionized water,

25 μL Phusion High-Fidelity PCR Master Mix (Thermo Fisher, Waltham, MA, USA),

2 μL forward primer (10 μM),

2 μL reverse primer (10 μM),

and 1 μL culture of S. pasteurii DSM33 as the template;

2) The PCR program:

a)   one cycle of 98°C for 10 min;

b)   30 cycles of 98°C for 10s, 55°C for 30s, and 72°C for 3 min;

c)   one cycle of 72°C for 5 min;

3) Run 1% gel electrophoresis to verify the size of amplified fragments;

4) Extract DNA from the gel using the TIANgel Midi Purification Kit (Tiangen, Beijing, China).

Notes & Results:

Succeeded.

 

Week 2 (7.15-7.19)

3. pET28a-Ure Construction:

1) Digest pET28a-OsMT1 with FastDigest NheI and XhoI (Thermo Fisher, Waltham, MA, USA);

2) Run 1% gel electrophoresis to verify the size of amplified fragments;

3) Extract DNA using the TIANgel Midi Purification Kit (Tiangen, Beijing, China);

4) Measure the purity and concentration of the DNA samples with a NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA, USA);

5) Use Vazyme ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, Jiangsu, China) to construct the recombinant vector pET28a-Ure:

6) Transform the recombinant vectors into Escherichia coli DH5α;

7) Screen on LB plates containing 50 μg/mL kanamycin;

8) Perform colony PCR for further screening;

9) Culture the positive transformants in liquid LB;

10)  Extract plasmids using the TIANprep Rapid Mini Plasmid Kit (Tiangen, Beijing, China);

11)  Confirm by DNA sequencing (GENEWIZ, Suzhou, Jiangsu, China).

Notes & Results:

Succeeded.

 

Week 3 (7.22-7.26)

4. pET28a-OsMT1-Ure Construction:

1) Linearize part pET28a-OsMT1 by PCR. This segment contains OsMT1 and half of the vector backbone;

2) Linearize part pET28a-Ure by PCR. This segment contains the urease gene cluster and the other half of the vector backbone;

3) Run 1% gel electrophoresis to verify the size of amplified fragments;

4) Extract DNA using the TIANgel Midi Purification Kit (Tiangen, Beijing, China);

5) Measure the purity and concentration of the DNA samples with a NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA, USA);

6) Use Vazyme ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, Jiangsu, China) to construct the recombinant vector pET28a-OsMT1-Ure:

7) Transform the recombinant vectors into E. coli DH5α;

8) Screen on LB plates containing 50 μg/mL kanamycin;

9) Perform colony PCR for further screening;

10)  Culture the positive transformants in liquid LB;

11)  Extract plasmids using the TIANprep Rapid Mini Plasmid Kit (Tiangen, Beijing, China);

12)  Confirm by DNA sequencing (GENEWIZ, Suzhou, Jiangsu, China).

Notes & Results:

We managed to get the linearized part pET28a-OsMT1 (4277 bp) by PCR but failed to get bands from pET28a-Ure (6956 bp) on our first try, probably due to reduced PCR efficiency when amplifying a long fragment. We tried to adjust the template amount but it did not help. After several failed PCRs, we tried touchdown PCR from 65°C to 50°C, and the PCR products showed clear bands.

 

Week 4 (7.29-8.2)

5. E. coli BL21(DE3) transformation:

1) Transform pET28a-OsMT1, pET28a-Ure, and pET28a-OsMT1-Ure into E. coli BL21(DE3);

2) Screen on LB plates containing 50 μg/mL kanamycin;

3) Perform colony PCR for further screening;

4) Culture the positive transformants in liquid LB;

Notes & Results:

Succeeded.

 

Week 5 (8.5-8.9)

6. Measurement of engineered E. coli function with calcium:

1) Prepare calcium chloride (5 M in water) stock solution. Sterilize using 0.22 µm filters (BKMAM, Changde, Hunan, China);

2) Add urea to liquid LB (Solarbio, Beijing, China) to reach a 25 g/L final concentration. Also, add calcium chloride to reach a 50 mM final concentration;

3) Aliquot 10 mL LB-urea-calcium media from Step 2) in each 50 mL centrifuge tube;

4) Inoculate the original and engineered E. coli into the centrifuge tubes;

5) Culture overnight at 37 ℃, 200 rpm;

6) On day 2, observe cultures under an MSD-S280 light microscope (Murzider, Dongguan, Guangdong, China) to see if calcium carbonate precipitated.

Notes & Results:

Results showed that only E. coli DH5α containing pET28a-Ure and pET28a-OsMT1-Ure facilitated calcium carbonate precipitation, as shown in the photo on the right. Interestingly, the E. coli BL21(DE3) containing the same vectors did not form carbonate precipitates, as shown in the photo on the left.

 

This result is unexpected, as we anticipated higher gene expression in BL21 compared to DH5α, which should result in greater urease activity and consequently more precipitate formation in BL21. To investigate what might have caused the gene to be non-functional in BL21, we decided to perform SDS-PAGE using cell lysates.

 

7. SDS-PAGE analysis of cell lysates

1) Incubate the original and engineered E. coli DH5α and BL21 overnight at 37 ℃;

2) Harvest cells by centrifugation;

3) Resuspend the cells in SDS Lysis Buffer (Beyotime, Shanghai, China) with protease inhibitor cocktail (Beyotime, Shanghai, China) added;

4) Add an equal volume of acid-washed glass beads (0.1 mm diameter);

5) Vortex the sample for 30 seconds, then place it on ice for 30 seconds. Repeat for 20 cycles;

6) Centrifuge at 13000 g for 10 min;

7) Incubate the supernatants with Omni-Easy Protein Loading Buffer (Epizyme, Shanghai, China) at 37 ℃ for 30 min;

8) Run SDS-PAGE with BeyoGel 10% Precast Gel (Beyotime, Shanghai, China);

Notes & Results:

In standard bacterial total protein SDS-PAGE protocols, cells are typically boiled for lysis, and protein samples are boiled to achieve denaturation. However, due to the heat sensitivity of membrane proteins (such as our fusion OsMT1), we avoided heating the samples. Instead, we opted for glass bead lysis on ice and incubation at 37°C.

 

Our SDS-PAGE results showed that although the engineered DH5α strains were functional in Test 6, no significant changes were observed when running total cell protein on the gel, as shown in the photo on the left. This is likely due to the naturally low protein expression levels in DH5α. The results also confirmed the high efficiency of the urease enzyme complex, as it demonstrated functionality despite the absence of visibly detectable protein in total protein SDS-PAGE.

 

In contrast, in BL21, we observed the presence of the introduced proteins, including ureC (the main subunit of the urease gene cluster) and the fusion protein OsMT1, confirming the high expression levels of these proteins.

 

Despite high expression levels, no ureolytic activity was observed in BL21. This may be due to the excessive expression rate of proteins in BL21. High expression likely overwhelmed the cell’s folding machinery, resulting in misfolded proteins or inclusion bodies, causing the proteins non-functional. This issue is especially prominent with large proteins like ureC (61.44 kDa). Conversely, DH5α’s lower expression levels may have allowed the proteins more time to fold correctly, preserving their functionality.

 

Although this issue could potentially be mitigated by adjusting culture conditions, such as lowering the temperature, we decided to give up BL21-based strains because we believe in stability. In real-world applications, we do not want our bacteria to lose function simply because they are under high-temperature conditions. Additionally, DH5α is superior in plasmid stability, which is advantageous for practical use.

 

Therefore, we plan to only use DH5α-based strains in future tests.

 

8. Measurement of growth of engineered E. coli:

1) Prepare 150 mL liquid LB media (Solarbio, Beijing, China) in each flask;

2) Inoculate the original and engineered E. coli DH5α into the flasks;

3) Culture at 37 ℃, 200 rpm;

4) On hours 0, 2, 4, 6, 8, 24, 28, and 32, measure the OD600s using a NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA, USA);

Notes & Results:

Results showed that all strains had a similar growth rate.

 

Week 6 (8.12-8.16)

9. Measurement of overnight heavy metal removal by engineered E. coli:

1) Prepare cadmium chloride, lead(II) nitrate, and mercury(II) nitrate (100 mM in water) stock solution. Sterilize using 0.22 µm filters (BKMAM, Changde, Hunan, China);

2) Add urea to liquid LB (Solarbio, Beijing, China) to reach a 25 g/L final concentration;

3) Aliquot 5 mL LB-urea media from Step 2) in each 15 mL centrifuge tube;

4) Add cadmium chloride, lead(II) nitrate, and mercury(II) nitrate respectively to reach a 0/0.0001/0.001/0.01/0.1/1/10 mM final concentration. There should be 76 tubes in total;

5) Inoculate the original and engineered E. coli DH5α into the centrifuge tubes;

6) Culture at 37 ℃, 200 rpm for 24 hours;

7) On day 2, measure the OD600s with an automated ELISA analyzer and 96-well plates;

8) Centrifuge the cultures and filter the supernatants with 0.22 µm filters (BKMAM, Changde, Hunan, China) to remove the bacteria and precipitates;

9) Measure the concentrations of heavy metal ions in the supernatants using the Inductively coupled plasma mass spectrometry (ICP-MS), conducted by Convinced-test Tech. Co., Ltd, Nanjiang, Jiangsu, China.

Notes & Results:

The results showed that our engineered bacteria significantly removed heavy metal ions from the solution within a certain range of initial concentrations.

 

At a very high concentration (10 mM), cadmium chloride and lead(II) nitrate inhibited bacterial growth and spontaneously formed a large amount of flocculent precipitate, as shown in the photo on the left. We suspect the precipitates to be Cd(OH)₂ and Cd(OH)Cl for cadmium, and Pb(OH)₂ and PbCl₂ for lead.

 

10. Measurement of heavy metal removal time by engineered E. coli:

1) Inoculate E. coli DH5α pET28a-OsMT1-Ure into 150 mL liquid LB (Solarbio, Beijing, China) in flasks;

2) Culture overnight at 37 ℃, 200 rpm;

3) Add urea to cultures to reach a 25 g/L final concentration;

4) Add cadmium chloride and lead(II) nitrate to cultures respectively to reach a 0.01/0.1 mM final concentration;

5) Culture at 37 ℃, 200 rpm;

6) On hours 0, 2, 4, 6, 8, and 10, collect 5 mL of the cultures;

7) Centrifuge the collected cultures and filter the supernatants with 0.22 µm filters (BKMAM, Changde, Hunan, China) to remove the bacteria and precipitates;

8) Measure the concentrations of heavy metal ions in the supernatants using the Inductively coupled plasma mass spectrometry (ICP-MS), conducted by Convinced-test Tech. Co., Ltd, Nanjiang, Jiangsu, China.

Notes & Results:

The results indicated that OsMT1 completed the reaction in 2 hours, whereas MICP required 4 to 6 hours.