iGEM 2024 — Team whhs-pro-china

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CONTENT

[WHHS-Pro-China iGEM Project: Summary of INP-PBP and PelB-PBP Engineering Strain Experiments (July 1 - July 30)](#a1)
[Project Objective:](#b1)
[Experimental Overview:](#b2)
[Key Findings:](#b3)
[Summary Timeline for 30 Days (July 1 - 30)](#a2)
[Daily record：](#a3)
[WHHS-Pro-China raw data](#a4)

WHHS-Pro-China iGEM Project: Summary of INP-PBP and PelB-PBP Engineering Strain Experiments (July 1 - July 30)

Project Objective:

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.

Experimental Overview:

Construction of INP-PBP and PelB-PBP Strains:

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.

Phosphate Adsorption Experiments:

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.

Optimization of Adsorption Conditions:

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).

Phosphate Desorption Experiments:

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.

Component Analysis of Engineered Bacteria:

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.

Statistical Analysis:

All data were analyzed using GraphPad Prism. Statistically significant differences in phosphate adsorption were found between different pH and temperature conditions (p < 0.05).

Key Findings:

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.

Summary Timeline for 30 Days (July 1 - 30)

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|  |

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. |

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|  |

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. |

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|  |

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. |

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|  |

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. |

Daily record：

WHHS-Pro-China raw data

![](https://static.igem.wiki/teams/5449/description/top1.png)

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CONTENT

[Abstract](#a1)
[Cycle 1: INP-PBP](#a2)
[Design](#b1)
[Build](#b2)
[Test](#b3)
[Test 1: Phosphate Adsorption Capacity](#b4)
[Test 2: Effect of Phosphate Starvation on Adsorption Capacity](#b5)
[Test 3: Optimization of Adsorption Conditions (pH and Temperature)](#b6)
[Test 4: Phosphate Desorption Efficiency](#b7)
[Test 5: Verification of INP Anchoring of PBP on the Cell Membrane](#b8)
[Learn](#b9)
[Cycle 2: PelB-PBP](#a3)
[Design](#b10)
[Build](#b11)
[Test](#b12)
[Test 1: Phosphate Adsorption Efficiency of PelB-PBP](#b13)
[Test 2: Immobilized PBP Adsorption Capacity](#b14)
[Test 3: Optimization of pH and Temperature for Immobilized PBP Desorption](#b15)
[Test 4: Secretion Efficiency Analysis](#b16)
[Learn](#b17)
[Conclusion：](#a4)
[References：](#a5)

Abstract

This project applied engineering principles to develop Escherichia coli  strains for optimized phosphate recovery through two iterative cycles. In **Cycle 1**, phosphate-binding protein (PBP) was anchored to the cell surface via ice nucleation protein (INP). The **Design** phase involved creating a strategy to display PBP on the cell membrane, while the **Build** phase focused on codon optimization and plasmid construction to achieve surface display. **Testing** confirmed that surface-displayed PBP significantly improved phosphate adsorption, with optimal conditions identified at pH 7 and 35°C. Phosphate desorption was most efficient under extreme pH (3 and 10) and elevated temperatures (45°C). Through **Learning,** phosphate starvation was found to further enhance adsorption capacity. In **Cycle 2,** PBP was secreted extracellularly using the PelB signal peptide. The **Design** targeted extracellular secretion, while **Build** facilitated PBP production and secretion. **Testing** of immobilized PBP on NHS-activated beads demonstrated superior adsorption and desorption performance under extreme conditions. **Learning** emphasized the need to optimize secretion efficiency and strain stability. These cycles of design, building, testing, and iterative learning provide a robust foundation for scalable phosphate recovery systems.

Cycle 1: INP-PBP

Design

In Cycle 1, the goal was to design an E. coli  strain that could efficiently recover phosphate by anchoring the phosphate-binding protein (PBP) on the cell surface. The core concept was to use ice nucleation protein (INP) as a membrane anchor, allowing PBP to be displayed on the cell membrane and thus exposed to the external environment for direct phosphate adsorption.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img1.png)

Figure 1.Cycle 1 diagram

Build

Once the design strategy was established, the next step was to build the INP-PBP system. The following steps were executed to construct the engineered strain:

**Gene synthesis and codon optimization:** The coding sequence for PstS (phosphate-binding protein) was synthesized. Additionally, a truncated version of the ice nucleation protein (INP) was used to ensure efficient membrane anchoring. Both sequences were codon-optimized for E. coli  to enhance expression efficiency.

**Plasmid construction:** The PBP gene was fused with the INP sequence at the N-terminus to create a single fusion protein. The genes were inserted into the pET23b vector via NdeI and XhoI restriction sites, under the control of a T7 promoter. This allowed for constitutive expression of the INP-PBP fusion protein in E. coli .

**Transformation and expression:** The recombinant plasmid was transformed into E. coli  DH5α for plasmid storage and then into E. coli  BL21 for protein expression. Both strains were grown in LB medium containing ampicillin (50 μg/mL) at 37°C. E. coli  DH5α was used solely for plasmid storage, while BL21 was utilized for expression due to its high protein production capability.

**Verification of protein expression:** To confirm the successful construction and expression of the INP-PBP protein, gel electrophoresis was performed after protein induction. This step verified that the fusion protein had the correct molecular weight, ensuring that the INP-PBP system was properly built.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img2.png)

Figure 2. Gel electrophoresis analysis of INP, PBP, and INP-PBP constructs.

Test

Several tests were conducted to evaluate the performance of the INP-PBP system. The goal was to verify the phosphate adsorption capacity of the engineered bacteria under different conditions.

Test 1: Phosphate Adsorption Capacity

**Objective:** To compare the phosphate adsorption efficiency between the engineered INP-PBP strain and a control strain expressing PBP intracellularly.

**Method:**

Both the experimental group (INP-PBP) and the control group (intracellular PBP) were cultured in LB medium containing 50 μg/mL ampicillin at 37°C and 250 rpm for 12 hours.

After culturing, the cell density was adjusted to OD600 = 1. Phosphate (KH₂PO₄) was added at a concentration of 10 mg/L, and the mixture was incubated at room temperature for 3 hours with shaking.

The remaining phosphate concentration in the solution was measured using the **Malachite Green Phosphate Detection Kit.** This kit forms a green phosphomolybdic acid complex when inorganic phosphate is present, and the absorbance was measured at 630 nm.

**Principle of Malachite Green Phosphate Detection Kit:**

Malachite Green Phosphate Detection Kit combines Malachite Green and Molybdate with free inorganic phosphate in solution. The green phosphomolybdic acid complex is formed. This complex has a characteristic absorbance in the wavelength range 620-640 nm, and the intensity of the absorbance is proportional to the phosphate concentration in the solution. By measuring the absorbance at 630 nm, the residual phosphate concentration in the solution can be quantitatively analyzed.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img3.png)

Figure 3. Phosphate adsorption efficiency of INP-PBP bacteria compared to intracellular PBP expression.

**Results:**

The engineered INP-PBP strain significantly reduced the phosphate concentration in the solution, indicating that the surface-displayed PBP effectively adsorbed phosphate.In contrast, the control strain with intracellular PBP showed no significant reduction in phosphate concentration, confirming that surface display is necessary for effective adsorption.

PBP must be displayed on the cell surface to efficiently adsorb phosphate. Intracellular expression alone is insufficient for effective phosphate removal. This result highlights the importance of surface-displayed PBP for developing a high-efficiency phosphate recovery system.

Test 2: Effect of Phosphate Starvation on Adsorption Capacity

**Objective:** To determine how phosphate starvation impacts the phosphate adsorption performance of the INP-PBP strain.

**Method:**

Two groups of INP-PBP bacteria were used: one subjected to phosphate starvation and the other without starvation. Both groups were then tested for phosphate adsorption using the Malachite Green method.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img4.png)

Figure 4: Effect of phosphate starvation on phosphate adsorption.

**Results:**

The phosphate-starved group exhibited a significantly higher adsorption capacity compared to the non-starved group.

Phosphate starvation further enhanced the adsorption capacity of INP-PBP engineered bacteria, demonstrating the effectiveness of genetic modification and starvation treatment in optimizing phosphate recovery.

Test 3: Optimization of Adsorption Conditions (pH and Temperature)

**Objective:** To identify the optimal pH and temperature conditions for phosphate adsorption by INP-PBP bacteria.

**Method:**

The engineered bacteria were incubated in solutions with varying pH values (3, 5, 7, 8, 10) and temperatures (25°C, 35°C, 45°C). After incubation, the remaining phosphate concentration in each solution was measured using the Malachite Green method.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img5.png)

Figure 5: Optimal pH and temperature for phosphate adsorption by INP-PBP bacteria.

**Results:**

The highest adsorption efficiency was observed at pH 7 and 35°C.

Optimal phosphate adsorption by INP-PBP bacteria occurred at pH 7 and 35°C, making these ideal conditions for the phosphate recovery system.

Test 4: Phosphate Desorption Efficiency

**Objective:** The phosphate desorption process is a key step in the whole phosphate recovery system. By adjusting the pH value and temperature of the environment, the engineered bacteria can release the phosphate that has been adsorbed, thus achieving the recovery of phosphate. Therefore, verifying the desorption capacity of engineered bacteria at different pH and temperature conditions is crucial for optimizing phosphate recovery systems.

**Method:**The INP-PBP bacteria were subjected to different pH (3, 5, 7, 8, 10) and temperature (25°C, 35°C, 45°C) conditions, and the amount of phosphate released from the bacteria was measured.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img6.png)

Figure 6: Phosphate desorption efficiency under various pH and temperature conditions.

**Results:**

Desorption was most efficient under acidic (pH 3) and alkaline (pH 10) conditions, and at 45°C. Strong acidic and alkaline conditions, along with high temperatures, promote effective phosphate desorption, providing crucial parameters for optimizing phosphate recovery systems.

Test 5: Verification of INP Anchoring of PBP on the Cell Membrane

**Objective：**This test aimed to verify if INP successfully anchored PBP to the cell membrane by comparing the phosphate adsorption capacities of cell membrane and cytoplasmic fractions.

**Experimental Setup：**

• **Bacterial Culture: I**NP-PBP bacteria were cultured overnight in LB medium (50 mL) at 37°C.

• **Cell Lysis:** After harvesting the cells, they were resuspended in PBS and lysed using ultrasonication (150W, 1-second pulse, 3-second interval, 20 minutes).

• **Fraction Separation:** The lysate was first centrifuged at 5,000 rpm for 10 minutes. The supernatant was then ultracentrifuged at 39,000 rpm for 1 hour, separating the cytoplasmic fraction (supernatant) and cell membrane fraction (pellet).

• **Resuspension:** The cell membrane pellet was resuspended in 2 mL PBS for adsorption experiments.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img7.png)

Figure 7: Phosphate adsorption capacity of cell membrane versus cytoplasmic fractions.

**Results**

The **cell membrane fraction** exhibited significantly higher phosphate adsorption than the cytoplasmic fraction, indicating that PBP was primarily located on the membrane.This confirmed that INP successfully anchored PBP to the cell membrane, enhancing phosphate adsorption.

The results validated the INP anchoring strategy, showing that PBP was effectively localized on the cell membrane, improving the bacteria’s phosphate adsorption efficiency.

Learn

Through the series of tests conducted in Cycle 1, the INP-PBP system demonstrated effective phosphate adsorption, particularly when PBP was anchored on the cell surface. However, several challenges and limitations were identified, which highlighted the need for further improvements to enhance the practical applicability of the system:

**Survival in Complex Environments:** While the INP-PBP bacteria effectively adsorbed phosphate under controlled lab conditions, their survival rate in real-world environments, such as complex wastewater, was low. The outer membrane pressure in such environments appears to strain the engineered bacteria, reducing their viability and overall system efficiency.

**High Maintenance Costs:** Maintaining the activity and stability of the engineered bacteria was costly. Continuous operation of the system requires careful control of environmental conditions to prevent protein inactivation. This introduces additional complexity and raises the operational costs, making large-scale implementation more challenging.

**Limited Adsorption Capacity:** Although the surface display of PBP was effective, the overall phosphate adsorption capacity remained limited. Since PBP could only be displayed on the cell surface, the amount of protein available for phosphate binding was restricted by the surface area of the bacteria. This limitation makes it difficult to meet the demands of large-scale phosphate removal systems, especially in environments with high phosphate concentrations.

These challenges suggest that while the PhosLock 1.0 system validates the feasibility of phosphate recovery, it needs significant optimization. Future iterations should focus on improving bacterial robustness in complex environments, reducing operational costs by enhancing strain stability, and increasing adsorption capacity through alternative strategies, such as extracellular secretion or immobilization techniques. These insights directly informed the design of Cycle 2, where extracellular secretion of PBP was explored to overcome the limitations observed in this phase.

Cycle 2: PelB-PBP

Design

In Cycle 2, the design strategy shifted from displaying PBP on the cell surface to secreting it extracellularly. The primary goal was to increase the phosphate-binding protein (PBP) available for adsorption by releasing it into the extracellular space, allowing for more flexible application in large-scale systems. This strategy also aimed to address the limitations of Cycle 1, including the restricted adsorption capacity and the challenges of maintaining the engineered bacteria in harsh environments.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img8.png)

Figure 8.PhosRegen 2.0 gene circuit map

Key design considerations included:

**Extracellular Secretion:** To achieve efficient secretion, the PelB signal peptide was chosen to direct the PBP (PstS) to the periplasmic space, where it could be secreted into the extracellular medium.

**Protein Immobilization:** Once secreted, PBP would be immobilized on NHS-activated beads to increase its phosphate adsorption capacity and stability, ensuring efficient use in recovery systems.

**Scalability and Stability:** The design sought to overcome the survival challenges faced by engineered bacteria in complex wastewater environments by decoupling phosphate adsorption from bacterial viability. Immobilizing secreted PBP would also reduce the need for live bacteria to remain active throughout the process.

Build

Following the design, the construction of the PelB-PBP system proceeded with several critical steps:

**Gene Synthesis and Signal Peptide Fusion:** The coding sequence for PBP (PstS) was synthesized and fused with the PelB signal peptide, which targets proteins for secretion into the extracellular space. Both sequences were codon-optimized for expression in E. coli  .

**Plasmid Construction:** The PelB-PBP gene construct was inserted into the pET23b vector, with expression driven by the T7 promoter. This allowed for controlled expression of the fusion protein. The plasmid was transformed into E. coli  DH5α for storage and BL21 for protein expression.

**Verification of Expression:** The secreted PBP was confirmed through gel electrophoresis after bacterial culturing and induction with IPTG, ensuring that the PelB signal was correctly guiding PBP secretion.

**Immobilization of PBP:** Once secreted, the PBP was incubated with NHS-activated Sepharose beads. These beads covalently bind proteins via their primary amine groups, stabilizing the PBP on the bead surface, ready for phosphate adsorption.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img9.png)

Figure 9. Gel electrophoresis analysis of the PelB-PBP construct.

Test

To evaluate the performance of the PelB-PBP system, multiple tests were conducted focusing on adsorption capacity, secretion efficiency, and desorption performance.

Test 1: Phosphate Adsorption Efficiency of PelB-PBP

**Objective:** To assess the phosphate adsorption capacity of the secreted PBP, compared to non-engineered strains and intracellular PBP expression.

**Method:**

Three bacterial strains were tested: non-engineered E. coli  (negative control), E. coli  expressing intracellular PBP (control), and E. coli  secreting PBP via PelB.

Each strain was cultured in LB medium containing ampicillin at 250 rpm and 37°C for 12 hours. After culture, 1 mL of bacterial suspension was centrifuged to remove the supernatant and resuspended in Tris-HCl buffer.The cell density was adjusted to OD600 = 1, and 10 mg/L KH₂PO₄ was added to the solution.The reaction was conducted at room temperature under 250 rpm shaking for 3 hours, and phosphate concentration was measured using the Malachite Green Phosphate Detection Kit.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img10.png)

Figure 10: Phosphate adsorption efficiency comparison between non-engineered strains, intracellular PBP-expressing strains, and PelB-PBP strains.

**Results:**

The PelB-PBP strain exhibited the highest phosphate adsorption efficiency, significantly surpassing both the non-engineered and intracellular PBP-expressing strains. This confirmed that extracellular secretion enhanced the availability of PBP for phosphate binding.

Test 2: Immobilized PBP Adsorption Capacity

**Objective:** To measure the adsorption performance of PelB-PBP immobilized on NHS-activated beads.

**Method:**

To evaluate the adsorption and desorption efficiency of immobilized phosphate-binding proteins (PBP), crude enzyme extracts were obtained from engineered BL21 bacteria overexpressing PelB-PstS (with a C-terminal His tag). PBP was immobilized onto NHS-activated Sepharose 4 Fast Flow beads by incubating the enzyme with the NHS beads at 4°C for 16 hours. After multiple washes, the immobilized PBP beads were used in a reaction system with KH₂PO₄ to measure their phosphate adsorption capacity.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img11.png)

Figure 11: Phosphate adsorption capacity of free PelB-PstS enzyme versus immobilized PBP on NHS beads.

**Results:**

The immobilized PBP demonstrated significantly higher phosphate adsorption capacity compared to the free PBP in solution, confirming that immobilization not only stabilized the protein but also enhanced its functionality in phosphate recovery.

Test 3: Optimization of pH and Temperature for Immobilized PBP Desorption

**Objective:** To optimize the conditions for phosphate desorption from the immobilized PBP to allow for efficient phosphate recovery.

**Method:**

To investigate the effect of pH and temperature on phosphate desorption by immobilized phosphate-binding proteins (PBP), PelB-PstS was immobilized onto NHS-activated Sepharose beads. The beads were saturated with 150 mg/L KH₂PO₄, and after washing with Tris-HCl buffer, unbound phosphate was removed. Desorption was measured under different pH conditions (pH 3, 5, 7, 8, and 10) and temperatures (25°C, 35°C, and 45°C).

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img12.png)

Figure 12: Phosphate desorption efficiency under various pH and temperature conditions.

**Results:**

The optimal desorption occurred at pH 3 and pH 10, with higher temperatures (45°C) further enhancing the desorption efficiency. This suggests that extreme pH conditions and elevated temperatures are ideal for releasing phosphate from immobilized PBP, making it more suitable for controlled recovery systems.

Test 4: Secretion Efficiency Analysis

**Objective:** To assess the secretion efficiency of PelB-PBP and compare the relative amounts of PBP in the extracellular, periplasmic, and intracellular fractions.

**Method:**

After growing the PelB-PBP strain, the periplasmic and extracellular fractions were separated via cold osmotic shock, while the intracellular fraction was isolated by sonication. Phosphate adsorption in each fraction was measured to determine the location of PBP.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img13.png)

Figure 13: Phosphate adsorption capacity of total lysate, soluble fraction, and periplasmic fraction in PelB-PBP strains.

**Results:**

Contrary to expectations, the majority of PBP was retained inside the cell, particularly in the intracellular fraction, indicating that the secretion efficiency of the PelB signal peptide was limited.Only a small portion of PBP was found in the periplasmic and extracellular fractions, suggesting that while the PelB signal peptide did facilitate some secretion, it was not fully effective.

Learn

• **Limited Secretion Efficiency:** The results from Test 4 showed that most of the PBP remained inside the cell, with only a small fraction successfully secreted into the extracellular space. This suggests that the PelB signal peptide’s secretion efficiency needs significant improvement. Future efforts should focus on optimizing the signal peptide or exploring alternative secretion systems to ensure more efficient release of PBP.

• **Increased Adsorption Potential with Immobilization:** Despite the limited secretion, the small amount of extracellular PBP that was immobilized on NHS-activated beads demonstrated high phosphate adsorption efficiency. This confirmed that immobilization is a valuable strategy, even though the quantity of secreted PBP was lower than expected.

• **Decoupling from Bacterial Viability:** The immobilization of PBP, though limited by secretion, still offers an approach to decouple phosphate adsorption from the viability of the bacteria. This reduces the dependency on maintaining live bacteria in harsh conditions, which was a challenge in Cycle 1.

• **Optimization Needed for Industrial Scale:** Although extracellular secretion showed promise, the low secretion efficiency limits the scalability of the system. Improvements in both secretion and immobilization are necessary to make the system viable for large-scale phosphate recovery.

Conclusion：

This project successfully developed two engineered phosphate recovery systems through iterative cycles of **Design, Build, Test, and Learn.** In **Cycle 1,** PBP was anchored to the cell surface via INP, which improved phosphate adsorption under optimal conditions. However, the limited adsorption capacity and high maintenance requirements led to the system being less suitable for large-scale applications. In **Cycle 2,** PBP was secreted extracellularly using the PelB signal peptide. Although secretion efficiency was lower than expected, the small amount of secreted PBP that was immobilized on NHS-activated beads demonstrated high adsorption and desorption capacity. This strategy decoupled phosphate recovery from the viability of the bacteria, making the system more adaptable and scalable for industrial use.

Moving forward, the key focus should be on improving secretion efficiency to increase the availability of extracellular PBP, enhancing the immobilization strategy, and optimizing the system for industrial scalability. These improvements will help develop a more effective and practical phosphate recovery solution for real-world applications.

References：

[1]Poirier Y, Jaskolowski A, Clúa J. Phosphate acquisition and metabolism in plants. Curr Biol. 2022 Jun 20;32(12):R623-R629. doi: 10.1016/j.cub.2022.03.073. PMID: 35728542.

[2]Ali EM, Khairy HM. Environmental assessment of drainage water impacts on water quality and eutrophication level of Lake Idku, Egypt. Environ Pollut. 2016 Sep;216:437-449. doi: 10.1016/j.envpol.2016.05.064. Epub 2016 Jun 17. PMID: 27321880.

[3]Cho H, Bouain N, Zheng L, Rouached H. Plant resilience to phosphate limitation: current knowledge and future challenges. Crit Rev Biotechnol. 2021 Feb;41(1):63-71. doi: 10.1080/07388551.2020.1825321. Epub 2020 Oct 7. PMID: 33028118.

[4]Gonzalez, D., Richez, M., Bergonzi, C. et al. Crystal structure of the phosphate-binding protein (PBP-1) of an ABC-type phosphate transporter from Clostridium perfringens. Sci Rep 4, 6636 (2014). https://doi.org/10.1038/srep06636

[5]Diaz R, Mackey B, Chadalavada S, Kainthola J, Heck P, Goel R. Enhanced Bio-P removal: Past, present, and future - A comprehensive review. Chemosphere. 2022 Dec;309(Pt 2):136518. doi: 10.1016/j.chemosphere.2022.136518. Epub 2022 Sep 30. PMID: 36191763.

[6]Vikrant K, Kim KH, Ok YS, Tsang DCW, Tsang YF, Giri BS, Singh RS. Engineered/designer biochar for the removal of phosphate in water and wastewater. Sci Total Environ. 2018 Mar;616-617:1242-1260. doi: 10.1016/j.scitotenv.2017.10.193. Epub 2017 Oct 26. PMID: 29107379.

[7]Zahed MA, Salehi S, Tabari Y, Farraji H, Ataei-Kachooei S, Zinatizadeh AA, Kamali N, Mahjouri M. Phosphorus removal and recovery: state of the science and challenges. Environ Sci Pollut Res Int. 2022 Aug;29(39):58561-58589. doi: 10.1007/s11356-022-21637-5. Epub 2022 Jul 3. PMID: 35780273.

[9]Gonzalez, D., Richez, M., Bergonzi, C. et al. Crystal structure of the phosphate-binding protein (PBP-1) of an ABC-type phosphate transporter from Clostridium perfringens. Sci Rep 4, 6636 (2014). https://doi.org/10.1038/srep06636

[10]Kinetics, Affinity, Thermodynamics, and Selectivity of Phosphate Removal Using Immobilized Phosphate-Binding Proteins ,Kaushik Venkiteshwaran, Erin Wells, and Brooke K. Mayer,Environmental Science & Technology 2020 54 (17), 10885-10894,DOI: 10.1021/acs.est.0c02272

[11]Brooke K Mayer, Justin M Hutchison, Eric S McLamore, Maria Torres, Kaushik Venkiteshwaran,Phosphate-binding proteins and peptides: from molecular mechanisms to potential applications,Current Opinion in Biotechnology,Volume 90,2024,103199,ISSN 0958-1669,https://doi.org/10.1016/j.copbio.2024.103199.

[12]Hicham Amar, Mostafa Benzaazoua, Abdellatif Elghali, Rachid Hakkou, Yassine Taha,Waste rock reprocessing to enhance the sustainability of phosphate reserves: A critical review,Journal of Cleaner Production,Volume 381, Part 1,2022,135151,ISSN 0959-6526,https://www.biorxiv.org/content/10.1101/830851v1.full

![](https://static.igem.wiki/teams/5449/description/top1.png)

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{% block title %}Experiments{% endblock %}
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CONTENT

[Construction of INP-PBP engineering strain](#a1)
[The phosphate adsorption capacity of INP-PBP engineering bacteria was analyzed](#a2)
[The optimum conditions of phosphate adsorption by INP-PBP engineering bacteria were analyzed](#a3)
[The phosphate desorption capacity of INP-PBP engineering bacteria was analyzed](#a4)
[Isolation of cytoplasmic and membrane components of INP-PstS engineering bacteria](#a5)
[Construction of PelB-PBP engineering strain](#a6)
[The phosphate adsorption capacity of PelB-PBP engineering bacteria was analyzed](#a7)
[Isolation of soluble and periplasmic components of PelB-PstS engineering bacteria](#a8)
[Western Blot analysis (to be done)](#a9)
[Immobilized PBP](#a10)
[The adsorption effect of immobilized PBP on Pi was tested](#a11)
[Statistical analysis](#a12)
[S0196 Malachite Green Phosphate Detection Kit：](#a13)
[NHS-activated Sepharose 4 Fast Flow：](#a14)

Construction of INP-PBP engineering strain

The coding gene sequence of phosphate-binding protein (PBPs) PstS was synthesized, and the ice-crystal nucleoprotein truncated sequence (INP) was synthesized and placed upstream of PstS. The above genes were codon optimized for E. coli, and then cloned into pET23b plasmid through NdeI and XhoI restriction sites to obtain the recombinant plasmid (Genewiz, USA). The recombinant plasmid expresses downstream coding genes through T7 promoter composition. After the sequencing was verified correctly (Cytology, China), the recombinant plasmid was extracted using the plasmid extraction kit (Tiangen, China). Next, the recombinant plasmid was transformed into E.coli DH5α and BL21. E.coli DH5α was used to store the plasmid and E.coli BL21 was used to express the plasmid. The engineered strains were cultured in LB medium containing ampicillin (Amp)(50 μg/mL) at 37°C.

The phosphate adsorption capacity of INP-PBP engineering bacteria was analyzed

The engineered strains were cultured in Luria-Bertani (LB) medium containing ampicillin (final concentration 50 μg/mL). The culture conditions were 250 rpm and 37°C. After 12 hours of culture, 1 mL bacterial solution was taken, centrifuged (5000 rpm, 10 min), the supernatant was abandoned, and the bacterial precipitate was suspended with Tris-HCl buffer (pH 7.4), and the cell density (OD600) was adjusted to 1. Add 10 mg/L KH2PO4. Reaction at room temperature at 250 rpm for 3 hours. The concentration of Phosphate in solution was analyzed using the Malachite Green Phosphate Detection Kit (S0196M, Beyotime).

The optimum conditions of phosphate adsorption by INP-PBP engineering bacteria were analyzed

To analyze the effect of phosphorus starvation on the adsorption capacity of engineered bacteria. Before the adsorption experiment, the engineered bacteria (OD600 = 1) were suspended in Tris-HCl buffer (pH 7) and reacted at 250 rpm at room temperature for 3 hours, so that the cells were placed under low phosphorus conditions, and then the adsorption experiment was carried out. In addition, the engineered bacteria (OD600 = 1) were re-suspended in buffers of different pH and treated under the same conditions for 3 hours. Where pH 3 and 5 are acetic acid buffers, pH 7 is PBS buffers, and pH 8 and 10 are Tris-HCl buffers. Then the phosphate adsorption experiment was carried out. To analyze the effect of temperature, adjust the reaction temperature at 25°C, 35°C, or 45°C during the adsorption reaction.

The phosphate desorption capacity of INP-PBP engineering bacteria was analyzed

Bacteria were cultured overnight in LB medium containing a high concentration of Pi medium (150 mg/L KH2PO4). Centrifuge to collect bacterial precipitates. Use a buffer (10 mM Tris-HCl, 1 mM MgCl₂, pH 7.5) to wash the bacterial precipitate twice to remove residual media. It is then re-suspended into the buffer and the bacteria OD600 = 1 is adjusted. Reaction at room temperature at 250 rpm for 3 hours. The concentration of Phosphate in solution was analyzed using the Malachite Green Phosphate Detection Kit (S0196M, Beyotime). The effect of pH on desorption was assessed using buffers of different pH values. The effect of temperature on desorption was evaluated using different reaction temperatures.

Isolation of cytoplasmic and membrane components of INP-PstS engineering bacteria

50 mL bacteria were cultured overnight, centrifuged (10000 rpm, 1 min) and then re-suspended with 10 ml PBS. Ultrasonic crushing (150W, ultrasonic 1 second, interval 3 seconds, a total of 20 minutes). Then centrifuge at 5000 rpm for 10min. The supernatant was collected and centrifuged at 39,000 rpm for 1h. The supernatant was collected as the cytoplasmic component. 2 mL PBS was used as the cell membrane component. The adsorption of phosphate by different components of engineering bacteria was determined according to the phosphate adsorption experiment process.

Construction of PelB-PBP engineering strain

The coding gene sequence of phosphate-binding protein (PBPs) PstS is synthesized, and the secretory tag (PelB) sequence is synthesized, which is placed upstream of PstS. The above genes were codon optimized for E. coli, and then cloned into pET23b plasmid through NdeI and XhoI restriction sites to obtain the recombinant plasmid (Genewiz, USA). The recombinant plasmid expresses downstream coding genes through T7 promoter composition. After the sequencing was verified correctly (Cytology, China), the recombinant plasmid was extracted using the plasmid extraction kit (Tiangen, China). Next, the recombinant plasmid was transformed into E.coli DH5α and BL21. E.coli DH5α was used to store the plasmid and E.coli BL21 was used to express the plasmid. The engineered strains were cultured in LB medium containing ampicillin (Amp)(50 μg/mL) at 37°C.

The phosphate adsorption capacity of PelB-PBP engineering bacteria was analyzed

The engineered strains were cultured in Luria-Bertani (LB) medium containing ampicillin (final concentration 50 μg/mL). The culture conditions were 250 rpm and 37°C. After 12 hours of culture, 1 mL of bacterial solution was taken, centrifuged (5000 rpm, 10 min), the supernatant was abandoned, and the bacterial precipitation was re-suspended with Tris-HCl buffer (pH 7.4), and the cell density (OD600) was adjusted to 1. Add 10 mg/L KH2PO4. Reaction at room temperature at 250 rpm for 3 hours. The concentration of Phosphate in solution was analyzed using the Malachite Green Phosphate Detection Kit (S0196M, Beyotime).

Isolation of soluble and periplasmic components of PelB-PstS engineering bacteria

Ten mL of the culture was collected and re-suspended on 1 mL of TES buffer (30mM Tris-HCl, 20% sucrose, and 1 mm EDTA; pH 8.0). The bacterial suspension was incubated at room temperature for 10 min and then centrifuged at 10,000×g and 4°C for 10 min. The precipitate was resuspended in 1 mL of cold 5 mM MgSO4 and incubated on ice for 10 minutes. Centrifuge again at 10,000×g and 4°C for 10 min and collect the supernatant as the periplasmic component. In order to collect the bacterial soluble components and the bacterial lysate, the bacterial lysate cultured overnight was centrifuged, re-suspended in 1 mL PBS, ultrasonic treatment (150W, ultrasonic 30 seconds, 2 times), and the bacterial lysate was collected. Then centrifuge at 10,000×g and 4°C for 10 min. The supernatant was collected as a soluble component. Then, the adsorption capacity of different components of engineering bacteria to phosphate was determined according to the adsorption experiment process.

Western Blot analysis (to be done)

Firstly, Bradford kit (Biyuntian) was used to determine the protein concentration of different components of engineered bacteria. The protein solution was then mixed with a 5× reduced protein sample buffer in a 4:1 ratio and denatured in a boiling water bath for 15 minutes. Protein was isolated at 120V using 15% SDS-PAGE. Subsequently, the proteins on the SDS-PAGE gel were transferred to the PVDF membrane under cold conditions. The membrane was closed with 5% skim milk (in TBST) at room temperature for 1 hour. Because pET23b carrier carries His label. The membrane was incubated overnight at 4°C with murine-derived His label antibodies (1:1000, AH367, Blue sky). Use TBST washing film three times for 10 minutes each time. They were then incubated at room temperature with horseradish peroxidase labeled Goat anti-mouse IgG (H+L) (1:1000 diluted, A0216, blue sky) for 1 hour. Use TBST washing film three times, after 10 minutes each time, mix the ECL A and B solution in a ratio of 1:1 and set aside. Place the washed PVDF film on absorbent paper and slightly dry the liquid on the surface of the film. The film is then placed on the chemiluminescent instrument rack and covered with the prepared ECL luminescent liquid to ensure that the film is completely immersed. After 1 minute of reaction, absorb the excess liquid at the top with absorbent paper, and put the film into the chemiluminescence instrument to start the chemiluminescence reaction.

Immobilized PBP

PelB-PstS (C-terminal with his tag) overexpressed engineering BL21 was cultured overnight in 100 mL LB medium. Centrifuge (10000 rpm, 1min) to collect bacterial precipitates. The binding buffer (10 mM imidazole, 20 mM Tris-HCl, 10 mM NaCl, pH = 7.4) was used to re-precipitate the suspended bacteria and adjust OD600 = 1. The bacterial solution was broken by ice bath ultrasonic wave. Before ultrasound, anhydrous ethanol was used to clean the ultrasonic broken cylinder head for 5min, and then distilled water was used to clean the cylinder head for 10min. The power was set at 150W, and the ultrasound was 1s at a interval of 2s. Centrifuge at 12,000 rpm at 4℃ for 30min, collect supernatant, discard precipitation, and obtain crude enzyme liquid samples. Subsequently, the PBP was fixed to the NHS-Activated Sepharose 4 Fast Flow (GE 17090601) bead. Specifically, fresh NHS beads (stored in 100% isopropyl alcohol) are washed with a 1 mM HCl solution. Then, 20 mL PBP was added to 20 mL of washed NHS beads and mixed at 30 rpm at 4°C for 16 hours. Beads fixed with PBP (PBP beads) were washed three times successively with 20 mL of 20 mM Tris-HCl (pH 7.5). The control beads are prepared following the same procedure as the PBP beads, but without the addition of PBP.

The adsorption effect of immobilized PBP on Pi was tested

0.25 mL of PBP beads, control beads or PBP enzyme solution were added to a 2 mL centrifuge tube. 1 mL reaction buffer (10 mM Tris-HCl, 1 mm MgCl2, 10 mg/L KH2PO4, pH 7.4) was added to the tube for 10 min at 25°C. Pi adsorption effect was measured.

The desorption effect of immobilized PBP on Pi was tested

Add 0.25 mL PBP beads to 2 mL centrifuge tube. Then 1 mL phosphate saturation buffer (10 mM Tris-HCl, 1 mM MgCl2, 150 mg/L KH2PO4, pH 7.4) was added to ensure the saturation adsorption of PBP beads on Pi. Mix gently and let stand at 25°C for 10 minutes. The PBP beads were then washed three more times with 1 mL buffer (10 mM Tris-HCl, 1 mM MgCl₂, pH 7.5) to remove the unbound Pi. Then, the Pi content was measured and the influence of different conditions on Pi desorption was evaluated. To assess the effects of temperature. Adjust the reaction temperature to 25°C, 35°C, or 45°C (pH 7.5). To assess the effect of pH, adjust the pH of the reaction buffer to the final values 3, 5, 7, 8, 10 (keep the temperature at 25°C) using HCl or NaOH to assess the effect of pH on Pi.

Statistical analysis

The data was analyzed and plotted using Graphpad Prism software. Data are presented as mean ± standard deviation (SD). For comparison of multiple sets of data, differences were analyzed using One-way ANOVA and Tukey post hoc tests. For the two sets of data, the two-tailed student's t-test was used for analysis. A P-value of less than 0.05 was considered statistically significant.

S0196 Malachite Green Phosphate Detection Kit：

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### S0196 Malachite Green Phosphate DetectionKit.pdf

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NHS-activated Sepharose 4 Fast Flow：

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### NHS-activated Sepharose 4 Fast Flow.pdf

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CONTENT

[Abstract](#a1)
[Construction of INP-PBP Engineered Strains](#a2)
[Phosphate Adsorption Capacity of INP-PBP Engineered Bacteria](#a3)
[Effect of Phosphate Starvation on Phosphate Adsorption Capacity](#a4)
[Optimization of Phosphate Adsorption Conditions for INP-PBP Bacteria](#a5)
[Phosphate Desorption Capacity of INP-PBP Bacteria](#a6)
[Verification of INP Anchoring of PBP on the Cell Membrane](#a7)
[Construction of PelB-PBP Engineered Strains](#a8)
[Phosphate Adsorption Capacity of PelB-PBP Engineered Bacteria](#a9)
[Effect of Soluble and Periplasmic Fractions on Phosphate Adsorption in PelB-PBP Engineered Strains](#a10)
[Phosphate Desorption Efficiency of Immobilized PBP](#a11)
[Effect of pH and Temperature on Phosphate Desorption by Immobilized PBP](#a12)
[Conclusion：](#a13)

Abstract

This study focuses on the application of phosphate-binding proteins (PBP) in phosphate recovery and explores optimization and validation in different engineered strains. First, **INP-PBP engineered bacteria **were constructed, where PBP was anchored to the cell membrane via ice nucleation protein (INP), and its phosphate adsorption capacity was verified. Additionally, the impact of phosphate starvation on the adsorption performance of INP-PBP bacteria was analyzed, alongside their desorption capacity under various pH and temperature conditions. Second, **PelB-PBP engineered bacteria** were developed, using the PelB signal peptide to secrete PBP extracellularly. The secretion efficiency and phosphate adsorption were assessed by separating soluble and periplasmic fractions. Lastly, by immobilizing PBP onto NHS-activated beads, we evaluated the adsorption capacity of the immobilized PBP and its desorption efficiency under different pH and temperature conditions. The results showed that both engineered strains demonstrated strong phosphate adsorption capacities, and the desorption performance was optimized using different strategies, providing effective solutions for phosphate recovery systems.****

Construction of INP-PBP Engineered Strains

The coding sequence for phosphate-binding proteins (PBPs) PstS was synthesized, along with the truncated ice nucleation protein (INP) sequence, placed upstream of PstS. Codon optimization for Escherichia coli was performed, and the genes were cloned into the pET23b plasmid via NdeI and XhoI restriction sites, generating the recombinant plasmid (Genewiz, USA). The recombinant plasmid, driven by the T7 promoter for constitutive expression of downstream genes, was verified through sequencing (Qingke, China) and extracted using a plasmid extraction kit (TianGen, China). The plasmid was then transformed into E. coli DH5α and BL21 strains. E. coli DH5α was used for plasmid storage, while BL21 was used for plasmid expression. The engineered strains were cultured in LB medium containing ampicillin (Amp) (50 μg/mL) at 37°C.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img1.png)

Figure 1. Gel electrophoresis analysis of INP, PBP, and INP-PBP constructs.

Phosphate Adsorption Capacity of INP-PBP Engineered Bacteria

**Experimental Design Overview:**

This experiment aimed to verify the phosphate adsorption capacity of different engineered strains. The experimental group used INP-PBP bacteria expressing PBP on the cell surface, while the control group consisted of bacteria expressing PBP intracellularly. The comparison of phosphate adsorption efficiency between these two groups was used to assess the necessity of displaying PBP on the cell surface.

**• Experimental Group (INP-PBP bacteria):** PBP was anchored to the cell surface by INP, enabling direct phosphate adsorption in the solution.

**• Control Group (Intracellular PBP expression):** PBP was expressed intracellularly, unable to directly contact the phosphate in the solution.

**Procedure:**

Both groups were cultured in LB medium containing 50 μg/mL ampicillin at 37°C, 250 rpm for 12 hours. After 12 hours, the cell density was adjusted to OD600 = 1, followed by the addition of 10 mg/L KH₂PO₄ as the phosphate source. The mixture was incubated at room temperature with shaking for 3 hours. The remaining phosphate concentration in the solution was measured using the Malachite Green Phosphate Detection Kit.

Principle of Malachite Green Phosphate Detection Kit:

Malachite Green Phosphate Detection Kit combines Malachite Green and Molybdate with free inorganic phosphate in solution. The green phosphomolybdic acid complex is formed. This complex has a characteristic absorbance in the wavelength range 620-640 nm, and the intensity of the absorbance is proportional to the phosphate concentration in the solution. By measuring the absorbance at 630 nm, the residual phosphate concentration in the solution can be quantitatively analyzed.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img2.png)

Figure 2. Phosphate adsorption efficiency of INP-PBP bacteria compared to intracellular PBP expression.

**Results:**

**• Experimental Group (INP-PBP bacteria):** The phosphate concentration in the solution significantly decreased, indicating that surface-displayed PBP efficiently adsorbed phosphate from the solution.

**• Control Group (Intracellular PBP expression):** Phosphate concentration remained unchanged, showing that intracellular PBP could not adsorb phosphate effectively.

**Conclusion:**

PBP must be displayed on the cell surface to efficiently adsorb phosphate. Intracellular expression alone is insufficient for effective phosphate removal. This result highlights the importance of surface-displayed PBP for developing a high-efficiency phosphate recovery system.

Effect of Phosphate Starvation on Phosphate Adsorption Capacity

**Experimental Setup:**

This experiment analyzed how phosphate starvation affected the phosphate adsorption capacity of engineered bacteria compared to non-engineered strains. The comparison between strains with and without phosphate starvation was used to evaluate the performance of the engineered bacteria.

**• Blue bars:** Non-engineered strains without phosphate starvation.

**• Red bars:** INP-PBP engineered bacteria, expressing PBP.

The remaining phosphate concentration in the solution was measured using the Malachite Green Phosphate Detection Kit to calculate the adsorption capacity.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img3.png)

Figure 3: Effect of phosphate starvation on phosphate adsorption.

**Results:**

**• Non-starved groups:** The non-engineered strain exhibited low adsorption capacity, while INP-PBP engineered bacteria showed significantly higher adsorption even without starvation.

**• Starved groups:** After phosphate starvation, the adsorption capacity of engineered bacteria increased dramatically.

**Conclusion:**

Phosphate starvation further enhanced the adsorption capacity of INP-PBP engineered bacteria, demonstrating the effectiveness of genetic modification and starvation treatment in optimizing phosphate recovery.

Optimization of Phosphate Adsorption Conditions for INP-PBP Bacteria

**Experimental Design:**

To determine optimal conditions for phosphate adsorption by INP-PBP bacteria, the experiment adjusted two variables: pH and temperature. The pH conditions tested were pH 3, 5, 7, 8, and 10, and the temperatures were 25°C, 35°C, and 45°C.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img4.png)

Figure 4: Optimal pH and temperature for phosphate adsorption by INP-PBP bacteria.

**Results:**

**• pH optimization:** Phosphate adsorption was highest at pH 7, with lower efficiency under acidic (pH 3 and 5) and alkaline (pH 8 and 10) conditions.

**• Temperature optimization:** Adsorption was most effective at 35°C, with decreased efficiency at both lower (25°C) and higher (45°C) temperatures.

**Conclusion:**

Optimal phosphate adsorption by INP-PBP bacteria occurred at pH 7 and 35°C, making these ideal conditions for the phosphate recovery system.

Phosphate Desorption Capacity of INP-PBP Bacteria

**Experimental Setup:**

The phosphate desorption process is a key step in the whole phosphate recovery system. By adjusting the pH value and temperature of the environment, the engineered bacteria can release the phosphate that has been adsorbed, thus achieving the recovery of phosphate. Therefore, verifying the desorption capacity of engineered bacteria at different pH and temperature conditions is crucial for optimizing phosphate recovery systems.

This experiment examined the phosphate desorption capacity of INP-PBP bacteria under different pH and temperature conditions to optimize phosphate recovery.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img5.png)

Figure 5: Phosphate desorption efficiency under various pH and temperature conditions.

**Results:**

Desorption was most efficient under acidic (pH 3) and alkaline (pH 10) conditions and at higher temperatures (45°C).

**Conclusion:**

Strong acidic and alkaline conditions, along with high temperatures, promote effective phosphate desorption, providing crucial parameters for optimizing phosphate recovery systems.

Verification of INP Anchoring of PBP on the Cell Membrane

**Experimental Setup:**

To verify whether INP successfully anchored PBP on the cell membrane, cell membrane and cytoplasmic fractions were separated, and their phosphate adsorption capacities were measured.

Experimental steps for separating cell membrane and cytoplasmic components:

Bacterial culture: The engineered bacteria were first cultured overnight in LB medium (50 mL medium).

Cell collection and resuspension: After culture, centrifuge at 10,000 rpm for 1 min to collect bacterial precipitates. The precipitate was re-suspended with 10 mL PBS to ensure uniform cell distribution.

Cell lysis: Breaking up cells by ultrasound. The crushing condition was 150W power, ultrasound for 1 second, interval of 3 seconds, cycle for 20 minutes to ensure complete cell lysis.

Preliminary centrifugation: The broken cell mixture is centrifuged at 5,000 rpm for 10 minutes and the supernatant is collected for further separation.

Ultracentricentrifugation: The collected supernatant is centrifuged at 39,000 rpm for 1 hour to separate the cytoplasmic components (supernatant) and cell membrane components (precipitate).

Re-suspension of cell membrane components: The collected cell membrane precipitates were re-suspended with 2 mL PBS and gently mixed in preparation for adsorption experiments.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img6.png)

Figure 6: Phosphate adsorption capacity of cell membrane versus cytoplasmic fractions.

**Results:**

The experimental results showed that the phosphate adsorption capacity of the cell membrane component (red column) was significantly higher than that of the cytoplasmic component (blue column), indicating that PBP was mainly located on the cell membrane and demonstrated its phosphate adsorption function here. This result is consistent with our hypothesis that INP successfully anchors PBP to the cell membrane, resulting in stronger adsorption capacity of the cell membrane components.

If PBP is not successfully anchored to the cell membrane, all of the PBP will remain in the cytoplasm, resulting in higher adsorption capacity in the cytoplasmic portion and lower adsorption capacity in the cell membrane portion. Therefore, the results of the experiment verified that INP successfully located PBP on the cell membrane, and the significant improvement of the adsorption capacity of the cell membrane was the embodiment of this anchoring effect.

**Conclusion:**

The results clearly validate the function of INP, that is, INP successfully anchors PBP to the cell membrane. This makes the membrane components of the engineered bacteria exhibit higher phosphate adsorption capacity, while the cytoplasmic components exhibit lower adsorption capacity. If PBP is not successfully anchored to the membrane, only the cytoplasmic portion is expected to exhibit higher adsorption capacity. The results provide important support for the phosphate recovery system based on INP-PBP, indicating that this anchoring strategy is feasible and effective for improving the phosphate adsorption effect of engineered bacteria.

Although the PhosLock 1.0 system validates the effectiveness of phosphate removal, some problems have been encountered in practical applications. First of all, engineering bacteria have low survival rate in complex wastewater environment due to high outer membrane pressure, which affects system efficiency. Secondly, the cost of maintaining the activity and stability of the strain is high, and the complex operation and protein inactivation problems further increase the operating cost. Finally, phosphate-binding proteins can only be displayed on the cell surface, and the adsorption capacity is limited, which is difficult to meet the needs of large-scale wastewater treatment.

Construction of PelB-PBP Engineered Strains

To address the limitations observed with the INP-PBP system, the phosphate-binding protein (PBP) PstS coding sequence was synthesized, along with a secretion signal (PelB), which was placed upstream of the PstS gene. The genes were codon-optimized for E. coli and cloned into the pET23b plasmid using NdeI and XhoI restriction sites, creating a recombinant plasmid (Genewiz, USA). This plasmid was constitutively expressed under the T7 promoter. After sequence verification (Qingke, China), the plasmid was extracted using a plasmid extraction kit (TianGen, China). The recombinant plasmid was transformed into E. coli DH5α for plasmid storage and E. coli BL21 for expression. The engineered strains were cultured in LB medium containing ampicillin (50 μg/mL) at 37°C.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img7.png)

Figure 7. Gel electrophoresis analysis of the PelB-PBP construct.

After secretion, PBP was incubated with NHS beads. The NHS group forms stable peptide bonds with the primary amine groups of PBP, immobilizing it onto the magnetic beads, thus improving phosphate adsorption efficiency. This method is expected to enhance the operation efficiency and adsorption capacity of the PhosLock 1.0 system.

Phosphate Adsorption Capacity of PelB-PBP Engineered Bacteria

**Experimental Design Overview:**

To evaluate the phosphate adsorption capacity of different strains, the experiment involved three types of bacteria: non-engineered strains (blue bars), engineered strains expressing PBP intracellularly (red bars), and engineered strains secreting PBP with a PelB signal (green bars). Each strain was cultured in LB medium containing ampicillin at 250 rpm and 37°C for 12 hours. After culture, 1 mL of bacterial suspension was centrifuged to remove the supernatant and resuspended in Tris-HCl buffer. The cell density was adjusted to OD600 = 1, and 10 mg/L KH₂PO₄ was added to the solution. The reaction was conducted at room temperature under 250 rpm shaking for 3 hours, and phosphate concentration was measured using the Malachite Green Phosphate Detection Kit.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img8.png)

Figure 8: Phosphate adsorption efficiency comparison between non-engineered strains, intracellular PBP-expressing strains, and PelB-PBP strains.

**Results Explanation:**

The figure compares the phosphate adsorption capacity of different strains:

**• Blue bars:** Non-engineered strains exhibited low phosphate adsorption capacity but still demonstrated some adsorption ability, indicating that even non-engineered strains can adsorb phosphate to a certain extent under appropriate conditions.

**• Red bars:** Strains expressing PBP intracellularly performed better than non-engineered strains, but their adsorption capacity was limited since PBP was primarily expressed inside the cells and could not adequately interact with external phosphate.

**• Green bars:** Strains secreting PBP with the PelB signal showed the strongest phosphate adsorption capacity, suggesting that the secretion of PBP outside the cell significantly enhanced phosphate binding and adsorption efficiency.

**Conclusion:**

The results indicate that while non-engineered strains possess some inherent phosphate adsorption capacity, engineered strains, particularly those secreting PBP via the PelB signal, demonstrate significantly enhanced adsorption performance. This confirms that the strategy of secreting PBP extracellularly is effective in improving phosphate adsorption, providing a solid foundation for the development of efficient phosphate recovery systems.

Effect of Soluble and Periplasmic Fractions on Phosphate Adsorption in PelB-PBP Engineered Strains

**Experimental Design Overview:**

To assess the impact of different active fractions on phosphate adsorption in PelB-PBP engineered bacteria, the experiment separated the periplasmic and soluble fractions of the engineered strains. The soluble fraction was obtained by sonicating overnight cultures and centrifuging the lysate to collect the supernatant. The periplasmic fraction was isolated by resuspending the bacteria in TES buffer and performing cold treatment and centrifugation. Phosphate adsorption in each fraction was measured using the Malachite Green Phosphate Detection Kit.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img9.png)

Figure 9: Phosphate adsorption capacity of total lysate, soluble fraction, and periplasmic fraction in PelB-PBP strains.

**Results Explanation:**

The total lysate exhibited the strongest phosphate adsorption capacity, as it contained all cellular components, including the membrane-bound and intracellular PBP. The soluble fraction had a higher adsorption capacity than the periplasmic fraction, suggesting that not all PBPs were successfully secreted outside the cell. While the periplasmic fraction showed some adsorption, it was significantly lower than that of the soluble fraction, indicating that PBP had been secreted into the periplasmic space but with limited efficiency.

**Conclusion:**

The results indicate that although the PelB signal was able to secrete some PBP into the periplasmic space, a large portion of the PBP remained in the intracellular soluble fraction. This suggests that the secretion efficiency was limited, likely due to short culture times or insufficient secretion capacity of the PelB signal. Enhancing secretion efficiency through extended culture times or optimizing the signal peptide could further improve overall phosphate adsorption performance. These findings offer valuable insights for improving phosphate recovery systems.

Phosphate Desorption Efficiency of Immobilized PBP

**Experimental Design Overview:**

To evaluate the adsorption and desorption efficiency of immobilized phosphate-binding proteins (PBP), crude enzyme extracts were obtained from engineered BL21 bacteria overexpressing PelB-PstS (with a C-terminal His tag). PBP was immobilized onto NHS-activated Sepharose 4 Fast Flow beads by incubating the enzyme with the NHS beads at 4°C for 16 hours. After multiple washes, the immobilized PBP beads were used in a reaction system with KH₂PO₄ to measure their phosphate adsorption capacity.

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img10.png)

Figure 10: Phosphate adsorption capacity of free PelB-PstS enzyme versus immobilized PBP on NHS beads.

**Results Explanation:**

The figure shows significant differences in phosphate removal efficiency under different treatment conditions. The enzyme solution containing PelB-PstS demonstrated some phosphate adsorption capacity, but the immobilized PelB-PstS on NHS beads (PelB-PstS @ Bead) exhibited much higher phosphate removal efficiency. Immobilizing PBP onto the beads not only retained its function but also enhanced phosphate binding efficiency. In contrast, the control group (NHS beads alone) showed negligible phosphate adsorption.

**Conclusion:**

The results demonstrate that immobilizing PBP onto NHS-activated beads significantly enhances its phosphate adsorption capacity compared to the free enzyme solution. Immobilized PBP performs more efficiently under the same conditions. This finding validates the potential of immobilized PBP in phosphate recovery systems and lays a foundation for future applications in large-scale wastewater treatment.

Effect of pH and Temperature on Phosphate Desorption by Immobilized PBP

**Experimental Design Overview:**

To investigate the effect of pH and temperature on phosphate desorption by immobilized phosphate-binding proteins (PBP), PelB-PstS was immobilized onto NHS-activated Sepharose beads. The beads were saturated with 150 mg/L KH₂PO₄, and after washing with Tris-HCl buffer, unbound phosphate was removed. Desorption was measured under different pH conditions (pH 3, 5, 7, 8, and 10) and temperatures (25°C, 35°C, and 45°C).

![](https://static.igem.wiki/teams/5449/{{self.title()|lower}}/img11.png)

Figure 11: Phosphate desorption efficiency under various pH and temperature conditions.

**Results Explanation:**

The figure shows that both pH and temperature significantly affect the desorption efficiency of immobilized PBP. Desorption was most efficient under strong acidic (pH 3) and strong alkaline (pH 10) conditions, where phosphate was effectively released from the beads. Desorption efficiency was lowest under neutral conditions (pH 7). Furthermore, higher temperatures (45°C) significantly enhanced desorption efficiency, while desorption was weaker at lower temperatures (25°C).

**Conclusion:**

The results indicate that strong acidic (pH 3), strong alkaline (pH 10), and higher temperatures (45°C) significantly improve phosphate desorption by immobilized PBP, while neutral pH and lower temperatures hinder phosphate release. By adjusting pH and temperature conditions, the desorption process can be optimized, offering an effective means to control phosphate release and recovery in wastewater treatment systems.

Conclusion：

This study successfully constructed and optimized two engineered bacterial strains, verifying the roles of INP-PBP and PelB-PBP in phosphate adsorption. INP-PBP bacteria effectively anchored PBP to the cell membrane, exhibiting excellent adsorption performance under various conditions, especially enhanced by phosphate starvation. PelB-PBP bacteria, despite not fully secreting all PBP, still showed good phosphate adsorption efficiency. After immobilizing PBP onto NHS-activated beads, the phosphate desorption capacity was significantly improved, particularly under high temperatures and extreme pH conditions. Overall, the research confirms the feasibility of both INP-PBP and PelB-PBP strategies, providing a solid foundation for the further development of phosphate recovery systems.

![](https://static.igem.wiki/teams/5449/description/top1.png)

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