The goal of this experiment was to construct two engineered E. coli strains expressing phosphate-binding proteins (PBPs) fused to either an ice nucleoprotein (INP) or a secretory signal peptide (PelB). These strains were designed to enhance phosphate adsorption for potential use in environmental phosphate capture and recovery.
We synthesized the coding sequences for PBP PstS, and fused them with INP and PelB sequences. These were codon-optimized for E. coli and cloned into the pET23b plasmid.
The recombinant plasmids were transformed into E. coli DH5α (for storage) and BL21 (for expression).
Plasmid extraction and sequencing verification were conducted, confirming successful construction.
The adsorption capacity of the engineered INP-PBP and PelB-PBP strains was tested using Malachite Green Phosphate Detection Kit.
Bacterial cultures were adjusted to OD600=1, and phosphate adsorption was measured in a buffer containing 10 mg/L KH2PO4.
Results: Both engineered strains demonstrated significant phosphate adsorption, with INP-PBP showing slightly higher initial binding efficiency compared to PelB-PBP.
The phosphate adsorption capacity was optimized by testing different pH levels (3, 5, 7, 8, 10) and temperatures (25°C, 35°C, 45°C).
Results: Adsorption efficiency was highest at pH 7 and 35°C for both strains. Deviation from these conditions led to reduced adsorption, particularly at extreme pH values (pH 3 and pH 10).
Desorption studies were performed to assess how well the bacteria could release phosphate under varying pH and temperature conditions.
Results: Both strains showed effective phosphate release at pH 7.5, with desorption increasing with temperature up to 45°C.
We isolated the cytoplasmic and membrane components of INP-PBP and PelB-PBP strains to determine which bacterial components contributed most to phosphate adsorption.
Results: The membrane components exhibited the highest phosphate binding capacity, suggesting that membrane-bound PBP is the primary contributor to adsorption.
All data were analyzed using GraphPad Prism. Statistically significant differences in phosphate adsorption were found between different pH and temperature conditions (p < 0.05).
INP-PBP and PelB-PBP strains both exhibited strong phosphate adsorption capabilities, with INP-PBP showing marginally higher efficiency under optimal conditions.
Optimal conditions for phosphate adsorption were identified at pH 7 and 35°C.
Membrane components were found to play a crucial role in phosphate binding.
The desorption studies indicated effective phosphate release at higher temperatures and neutral pH, supporting the potential for reuse of the engineered strains.
| Date | Activity | Notes |
| July 1-3 | Prepare pET23b plasmid with INP-PBP and PelB-PBP gene constructs. Transform into E. coli DH5α and BL21for storage and expression. | Ensure successful transformation by conducting colony PCR and sequencing verification. |
| July 4-6 | Plasmid extraction from transformed E. coli strains (Tiangen extraction kit) and sequence verification. | Ensure plasmids are extracted from both DH5α and BL21 strains. |
| July 7-9 | Set up LB cultures for both INP-PBP and PelB-PBP engineering strains. Culture in LB + Amp (50 μg/mL). | Use LB broth to grow strains overnight at 37°C and 250 rpm for optimal growth. |
| July 10-12 | Perform phosphate adsorption experiment for INP-PBP strains. Adjust OD600 to 1, add KH2PO4, and incubate. Analyze phosphate concentration using Malachite Green Kit. | Follow the protocol for detecting phosphate adsorption, ensure replicates are included. |
| July 13-14 | Conduct phosphate desorption experiment for INP-PBP bacteria. Prepare bacterial cells, incubate in buffer, and analyze phosphate concentration. | Evaluate desorption under varying pH and temperatures. |
| July 15-17 | Perform phosphate adsorption experiment for PelB-PBP strains using similar conditions. | Same as INP-PBP phosphate adsorption experiment. Compare adsorption capacity across strains. |
| July 18-20 | Optimization of adsorption conditions for INP-PBP engineering strain by varying pH and temperature. Conduct adsorption tests with different buffers and temperatures. | Test phosphate adsorption under different pH (3, 5, 7, 8, 10) and temperatures (25°C, 35°C, 45°C). |
| July 21-23 | Perform optimization of adsorption conditions for PelB-PBP strains. Adjust buffer pH and incubation temperature to determine optimal adsorption conditions. | Follow similar pH and temperature variation protocol for PelB-PBP strains. |
| July 24-26 | Analyze cytoplasmic and membrane components of INP-PBP bacteria using ultracentrifugation. Test phosphate adsorption of the isolated components. | Separate components using ultracentrifuge and analyze phosphate binding of each component. |
| July 27-29 | Analyze soluble and periplasmic components of PelB-PBP bacteria. Extract these components and test adsorption ability. | Follow the extraction and phosphate binding protocol for PelB-PBP's components. |
| July 30 | Statistical analysis and troubleshooting. Perform data analysis and rerun key experiments if required. Prepare a summary report of the results and plan next steps if needed. | Analyze data using Graphpad Prism, check for significant results (p < 0.05). If necessary, repeat failed experiments or adjust protocols based on troubleshooting findings. |
