iGEM 2025 — Team yau-china

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### **Experimental Progress**

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### March

**March 12, 2025 – Project Initiation and Chassis Selection**

Objective: To officially initiate the project and select an appropriate bacterial strain as the chassis for subsequent experimental work.

Methods and Procedures: Potential bacterial strains were analyzed through comprehensive literature review, and their characteristics were verified using preliminary experiments. *E. coli* EC1000 was ultimately confirmed to support the normal replication of the pORI280 plasmid, meeting the requirements for use as a chassis strain.

Results: *E. coli* EC1000 was formally selected as the chassis strain for this project.

Observations and Records: *E. coli* EC1000 is a *lacZ*-deficient strain and does not express β-galactosidase; however, it contains the *gus* gene, which encodes β-glucuronidase.

Participants: Design Group, Literature Research Group

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**March 15, 2025 – *gus* Gene Background Verification**

Objective: To determine whether the endogenous *gus* gene in the EC1000 genome would interfere with subsequent reporter gene expression experiments.

Methods and Procedures: PCR molecular detection technology was employed in conjunction with phenotypic observation to comprehensively analyze the functional expression status of the *gus* gene in EC1000.

Results: Detection confirmed that the *gus* gene in EC1000 is functional, and its expression product may cause background signal interference in subsequent experiments.

Observations and Records: To eliminate background interference and achieve zero-background experimental conditions, it is necessary to knockout the *gus* gene in EC1000 to construct a zero-background chassis strain.

Participants: Chassis Modification Group

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**March 20, 2025 – Δ*gus* Gene Knockout**

Objective: To construct a zero-background chassis strain lacking the *gus* gene (*E. coli* EC1000Δ*gus*) and eliminate background interference from the endogenous *gus* gene in subsequent experiments.

Methods and Procedures:

A *gus* deletion (Δ*gus*) homologous recombination fragment was generated using PCR amplification.

Using the pDM4 suicide plasmid as the vector, conjugation transfer, single-crossover selection, and sucrose counter-selection were sequentially performed to achieve homologous recombination between the Δ*gus* fragment and the EC1000 genome.

Results: The *E. coli* EC1000Δ*gus* mutant strain was successfully obtained.

Observations and Records: Colony PCR molecular detection and phenotypic verification both confirmed the expected results, demonstrating complete deletion of the *gus* gene from the EC1000 strain.

Participants: Chassis Modification Group

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**March 28, 2025 – Sensor Module Construction**

Objective: To complete the construction of four sensor plasmids (Plasmid A) that will serve as core vectors for subsequent functional validation of the sensor-reporter system.

Methods and Procedures:

The four target genes—PqsR, SoxR, BrlR, and MexL—were individually inserted into expression cassettes driven by the Pcat promoter to construct the core sensor elements.

These core sensor elements were then transferred into different vectors, including pORI280, pDM4, pET28b, pUC18T-mini-TN7T, and p34SGm. A gentamicin (Gm) resistance gene was added to each vector to facilitate subsequent selection.

Results: Four sensor modules were successfully constructed.

Observations and Records: Four sensor modules (Plasmid A) meeting the design requirements were successfully created.

Participants: Molecular Cloning Group

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### April

**April 3, 2025 – Reporter Module Construction**

Objective: To construct reporter plasmids (Plasmid B) corresponding to the sensor plasmids (Plasmid A), thereby completing the vector framework for the four sensor-reporter systems.

Methods and Procedures:

Using mini-CTX as the vector backbone, a *lacZ* gene-based reporter system was constructed for signal detection.

Using pUC18T-mini-TN7T as the vector backbone, a *gus* gene-based reporter system was constructed to provide an additional signal detection pathway.

Results: The reporter plasmids (Plasmid B) were successfully constructed and matched with the previously created sensor plasmids (Plasmid A), forming four complete sensor-reporter plasmid sets.

Observations and Records: All constructed sensor-reporter plasmids are ready for immediate use in subsequent functional validation experiments.

Participants: Molecular Cloning Group

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**April 7, 2025 – Functional Validation and Failure Analysis**

Objective: To verify the functional effectiveness of the four sensor-reporter systems.

Methods and Procedures:

Experimental group: PQS (pyocyanin) or PYO (phenazine-1-carboxylic acid) was added to the system as an inducer.

Control group: Reporter plasmids lacking the response promoter were used to exclude non-specific signal interference.

Results:

The MexL-PphzA1 sensor-reporter system produced signals that showed no significant difference from the control group.

The control groups of the other three systems all exhibited obvious color changes or fluorescent signals.

Observations and Records:

The mini-CTX-*lacZ* vector is a high-copy plasmid in *E. coli*, which tends to result in high background signals.

In the pUC18T-mini-TN7T-*gus* vector, the Plac promoter can directly drive the expression of the downstream *gus* gene.

Participants: Functional Validation Group

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**Cycle 1 Summary (March 12 – April 7)**

Phase Achievements:

Chassis strain modification completed: Successfully constructed the zero-background chassis strain *E. coli* EC1000Δ*gus*, eliminating background interference from the endogenous *gus* gene.

Plasmid construction completed: Four sets of sensor plasmids (Plasmid A) and corresponding reporter plasmids (Plasmid B) were successfully constructed, forming complete sensor-reporter plasmid combinations.

Phase Issues:

Inappropriate selection of reporter vectors resulted in the failure of the four constructed sensor-reporter systems to achieve the expected functionality.

Subsequent Plans:

Screen and identify reporter vectors with low-background characteristics to replace the current vectors.

Reconstruct the sensor-reporter systems based on the new vectors and conduct functional validation experiments.

Timeframe: April 8 – May 9, 2025

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**April 8, 2025 – Vector Rescreening and Design**

Objective: To address the high-background issue of the reporter plasmids identified in Cycle 1 by screening and selecting new vectors with low-background characteristics, thereby laying the foundation for reconstructing the sensor-reporter system.

Methods and Procedures:

Through literature research, the characteristics of existing low-background vectors were analyzed. Combined with team discussions to assess feasibility, pET28b was ultimately selected as the new reporter plasmid backbone.

To ensure a single experimental variable, the structure and sequence of the original PAZ2-1 (sensor plasmid) were maintained unchanged, while continuing to use the chassis strain *E. coli* EC1000Δ*gus*.

Results:

pET28b was formally confirmed as the new reporter vector, and the relevant vector preparation work has been completed, with reconstruction of the sensor-reporter system set to begin.

Observations and Records:

pET28b is a classical expression vector commonly used in laboratories. Its intrinsic regulatory element characteristics theoretically result in low background signals, making it suitable for the current system optimization requirements.

Participants: Literature Research Group, Design Group

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**April 12, 2025 – Reporter Plasmid Construction (PpqsA-*lacZ*)**

Objective: To construct the PpqsA-*lacZ* reporter system in the pET28b vector to meet the requirements of low-background experiments.

Methods and Procedures:

The PpqsA promoter and *lacZ* gene fragment were ligated into the multiple cloning site (MCS region) of the pET28b vector.

Restriction enzyme digestion and DNA sequencing were performed to confirm the correctness of the cloning construct.

Results: The recombinant reporter plasmid pET28b-PpqsA-*lacZ* was successfully obtained.

Observations and Records: The cloning efficiency was high, and no abnormalities were observed during the experiment.

Participants: Molecular Cloning Group

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**April 14, 2025 – Reporter Plasmid Construction (PphzA1-*gus*)**

Objective: To construct the PphzA1-*gus* reporter system in the pET28b vector, supplementing the low-background reporter module portfolio.

Methods and Procedures:

The PphzA1 promoter and *gus* gene fragment were amplified using PCR technology.

The amplified fragments were ligated with the pET28b vector backbone.

Results: The recombinant reporter plasmid pET28b-PphzA1-*gus* was successfully obtained.

Observations and Records: DNA sequencing confirmed that the recombinant plasmid contained no base mutations and the sequence matched the design requirements.

Participants: Molecular Cloning Group

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**April 17, 2025 – Reporter Plasmid Construction (PbrlR-*gus*)**

Objective: To construct the PbrlR-*gus* reporter system in the pET28b vector, further completing the low-background sensor-reporter module library.

Methods and Procedures:

The PbrlR promoter and *gus* gene fragment were amplified using PCR technology.

The amplified fragments were ligated into the pET28b vector backbone to complete the recombinant plasmid construction.

Results: The recombinant reporter plasmid pET28b-PbrlR-*gus* was successfully obtained.

Observations and Records: The cloning efficiency was moderate, but the target plasmid was ultimately obtained successfully after optimization of the procedure.

Participants: Molecular Cloning Group

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**April 20, 2025 – Reporter Plasmid Construction (PmexG-*gus*)**

Objective: To construct the PmexG-*gus* reporter system in the pET28b vector, expanding the portfolio of low-background reporter systems.

Methods and Procedures:

The PmexG promoter and *gus* gene fragment were amplified using PCR technology.

The amplified fragments were inserted into the pET28b vector backbone to construct the recombinant reporter plasmid.

Results: The recombinant reporter plasmid pET28b-PmexG-*gus* was successfully obtained.

Observations and Records: DNA sequencing confirmed that the recombinant plasmid had the correct sequence with no base mutations or deletions.

Participants: Molecular Cloning Group

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**April 23, 2025 – Control Plasmid Construction**

Objective: To establish a control system lacking response promoters, providing a baseline for subsequent functional validation of the sensor-reporter systems and eliminating non-specific signal interference.

Methods and Procedures:

Construct the pET28b-*lacZ* control plasmid without a promoter.

Construct the pET28b-*gus* control plasmid without a promoter.

Results: Both control plasmids lacking response promoters were successfully constructed.

Observations and Records: These control plasmids serve as critical negative controls in functional validation experiments, ensuring the comparability of results between different experimental groups.

Participants: Molecular Cloning Group

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**April 28, 2025 – Plasmid Introduction and Test Strain Assembly**

Objective: To co-transform the sensor plasmids and reporter plasmids into the chassis strain, creating experimental strains ready for direct functional validation.

Methods and Procedures:

The four newly constructed reporter plasmids were each co-transformed with their corresponding PAZ2-1 sensor plasmids (containing PqsR, SoxR, BrlR, and MexL genes) into the chassis strain EC1000Δ*gus*.

The previously constructed control plasmids were simultaneously transformed into EC1000Δ*gus* to prepare control strains.

Results: All newly designed test strains and their corresponding control strains were successfully obtained.

Observations and Records: The co-transformation efficiency was high, and the strains exhibited good growth, making them suitable for subsequent functional validation.

Participants: Chassis Engineering Group

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### May

**May 2, 2025 – Functional Validation Experiment**

Objective: To verify whether the new system constructed on the pET28b vector resolves the high-background signal issue identified in Cycle 1.

Methods and Procedures:

Experimental group: Specific signaling molecules (PQS or PYO) were added to induce the test strains containing the corresponding sensor-reporter plasmids.

Control groups: Two types of controls were established: 1. Strains containing promoterless control plasmids. 2. Strains containing normal reporter plasmids but without the addition of signaling molecules.

Results: Significant background signals were still observed in all control groups.

Observations and Records: All control groups exhibited evident background signals, failing to achieve the expected low-background effect.

Participants: Functional Validation Group

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**May 6, 2025 – Failure Analysis Meeting**

Objective: The results of the current detection are consistent with the background signal issue observed in Cycle 1, indicating that the background interference phenomenon has recurred and that the current system still requires further optimization.

Methods and Procedures:

Conducted team discussions focusing on the structural features of the selected pET28b vector to identify potential effects of vector elements on reporter gene expression.

Performed a detailed examination of the pET28b plasmid map, with particular attention to the position and transcriptional direction of the KanR (kanamycin resistance) gene promoter, and evaluated its potential to interfere with downstream reporter genes.

Results: A key hypothesis was proposed — the KanR gene promoter in the pET28b vector may cause transcriptional crosstalk, leading to non-specific activation of the reporter genes (*lacZ*/*gus*) and subsequent generation of background signals.

Observations and Records: Combining the results from the two failures in Cycle 1 and Cycle 2, it can be concluded that the high-background problem is not solely due to inappropriate vector selection, but rather a common issue caused by specific structural elements within the vector, such as the resistance gene promoter.

Participants: Design Group, Signal Analysis Group

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**May 9, 2025 – Cycle 2 Phase Summary**

Objective: To review the current experimental progress and lessons learned from the failures in the two cycles, identify the core issues, and provide a basis for the redesign of the system in the next phase.

Results:

Reporter vector optimization failed again: The new sensor-reporter system constructed on the pET28b vector still did not resolve the high-background signal issue.

Root cause identified: Analysis confirmed that the core cause of the high background is likely "transcriptional crosstalk" triggered by internal vector elements (such as the resistance gene promoter).

Observations and Records:

Vector selection: In the next phase, vectors without endogenous promoters should be screened to minimize the risk of transcriptional crosstalk at the source.

Vector modification: If a suitable vector is difficult to find, redesigning the orientation of the resistance gene (reverse insertion) may be attempted to block its non-specific activation of the reporter gene.

Participants: The entire team

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**Cycle 2 Summary (April 8 – May 9, 2025)**

Core Achievements: Vector construction completed: Successfully reconstructed four sets of reporter plasmids and constructed corresponding control plasmids in parallel. No technical issues were encountered during the vector construction phase.

Key Issues:

System testing failed again: During functional validation of the newly constructed sensor-reporter systems, background signals remained significant and did not meet the expected results.

Potential cause identified: Through analysis and investigation, "transcriptional crosstalk" from the vector's resistance gene was proposed as a new potential cause of the high background signal.

Core Directions:

Core principle: The "transcriptional crosstalk" issue must be resolved, with the key pathway being the selection or modification of vectors.

Specific actions: Prioritize screening for vectors without endogenous promoters.

If screening yields no suitable candidates, modify existing vectors to eliminate interference from the resistance gene promoter.

Timeframe: May 10 – June 14, 2025

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**May 10, 2025 – New Vector Screening and Protocol Determination**

Objective: To fundamentally resolve the high-background issue caused by "transcriptional crosstalk" at the vector element level and provide a suitable vector for reconstructing the reporter module.

Methods and Procedures:

The structural characteristics of multiple candidate vectors were compared, and pMP220 was ultimately selected as the new reporter plasmid backbone based on the following criteria:

1. The multiple cloning site (MCS region) has a clean sequence without endogenous promoters, preventing non-specific transcriptional initiation.

2. The transcription direction of the resistance gene is opposite to that of the inserted fragment in the MCS region, effectively preventing "read-through" activation of the reporter gene by the resistance gene promoter.

Results: It was confirmed that pMP220 would be used for reconstructing subsequent reporter modules.

Observations and Records: This vector selection represents the first time that the transcriptional crosstalk issue has been addressed specifically from the perspective of "vector element design," rather than simply replacing the vector type.

Participants: Design Group, Signal Analysis Group

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**May 15, 2025 – Construction of PpqsA-*lacZ* Reporter System (pMP220)**

Objective: To reconstruct the PpqsA-*lacZ* reporter module in the pMP220 vector to meet the requirements of low-background experiments.

Methods and Procedures: The PpqsA promoter and *lacZ* gene fragment were amplified using PCR technology and ligated into the MCS region of the pMP220 vector.

Results: The recombinant reporter plasmid pMP220-PpqsA-*lacZ* was successfully obtained.

Observations and Records: DNA sequencing confirmed that the recombinant plasmid sequence was completely correct, with no base mutations or deletions.

Participants: Molecular Cloning Group

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**May 17, 2025 – Construction of PphzA1-*gus* Reporter System (pMP220)**

Objective: To reconstruct the PphzA1-*gus* reporter module in the pMP220 vector, further completing the low-background reporter system library.

Methods and Procedures: The PphzA1 promoter and *gus* gene fragment were amplified using PCR technology and inserted into the MCS region of the pMP220 vector.

Results: The recombinant reporter plasmid pMP220-PphzA1-*gus* was successfully obtained.

Observations and Records: DNA sequencing confirmed that the recombinant plasmid sequence was completely correct, with no base mutations or deletions.

Participants: Molecular Cloning Group

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**May 20, 2025 – Construction of PbrlR-*gus* Reporter System (pMP220)**

Objective: To reconstruct the PbrlR-*gus* reporter module in the pMP220 vector.

Methods and Procedures: The PbrlR fragment and *gus* fragment were separately amplified using PCR technology, and both fragments were inserted into the pMP220 vector through cloning techniques.

Results: The recombinant reporter plasmid pMP220-PbrlR-*gus* was successfully obtained.

Observations and Records: Restriction enzyme digestion confirmed that the recombinant plasmid sequence was correct and consistent with the experimental expectations.

Participants: Molecular Cloning Group

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**May 22, 2025 – Construction of PmexG-*gus* Reporter System (pMP220)**

Objective: To reconstruct the PmexG-*gus* reporter module in the pMP220 vector.

Methods and Procedures: The PmexG fragment and *gus* fragment were amplified using molecular biology techniques, and the two fragments were ligated together before being inserted into the pMP220 vector.

Results: The recombinant reporter plasmid pMP220-PmexG-*gus* was successfully constructed.

Observations and Records: Verification confirmed that the recombinant plasmid contained no base mutations, and the module construction was completed smoothly.

Participants: Molecular Cloning Group

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**May 25, 2025 – Construction of Control Plasmids (pMP220)**

Objective: To construct plasmids without response promoters to serve as negative controls for subsequent experiments.

Methods and Procedures: Recombinant plasmids pMP220-*lacZ* and pMP220-*gus* were constructed, both lacking the response promoter sequence.

Results: Two types of negative control plasmids, pMP220-*lacZ* and pMP220-*gus*, were successfully obtained.

Observations and Records: This batch of control plasmids will be used in subsequent functional validation experiments of the reporter modules to eliminate non-specific interference.

Participants: Molecular Cloning Group

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**May 28, 2025 – Test Strain Assembly**

Objective: To co-assemble the new reporter plasmids with the original sensor plasmids (PAZ2-1) into the chassis strain.

Methods and Procedures:

Co-transform the new reporter plasmids and the original sensor plasmids (PAZ2-1) into the chassis strain EC1000Δ*gus*.

Based on the differences in the transformed plasmids, establish and obtain both test groups and control groups.

Results: The assembly of the new round of test strains was successfully completed.

Observations and Records: The assembled strains showed normal colony growth without any abnormal morphology or growth retardation.

Participants: Chassis Engineering Group

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### June

**June 1, 2025 – Functional Validation Experiment**

Objective: To determine whether the new reporter system can effectively resolve the background signal interference issue.

Methods and Procedures:

Experimental group: PQS or PYO signaling molecules were added to the strain system containing the new reporter system.

Control group: A strain system containing only the empty vector was used to eliminate any signal interference from the vector itself.

Results:

Control group: No background signal was detected throughout the experiment.

Experimental group: After adding the signaling molecules, the strains exhibited the expected color/fluorescence response.

The new reporter system achieved normal functional operation for the first time.

Observations and Records: This experimental result represents a key breakthrough, directly demonstrating the scientific validity and feasibility of the new reporter system design.

Participants: Functional Validation Group

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**June 5, 2025 – Signal Stability Test**

Objective: To assess the stability and experimental reproducibility of the new reporter system.

Methods and Procedures:

Multiple repetitions of the new reporter system functional test experiment were performed.

Signal intensities of the strains were measured and recorded at different time points using different well plates to verify system stability.

Results:

Significant fluctuations in signal intensity were observed in the experimental group during the detection process.

There were substantial differences in the color/fluorescence intensity produced by the strains between different batches of repeated experiments.

Observations and Records: No background signal was detected in the control group, indicating that the background issue has been resolved. However, the signal stability and experimental reproducibility of the experimental group did not meet the expected standards.

Participants: Functional Validation Group

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**June 10, 2025 – Cause Analysis Meeting**

Objective: To analyze the reasons for the signal instability of the new reporter system.

Methods and Procedures:

Discussed the molecular characteristics and functional features of the vector (pORI280) used in PAZ2-1.

Evaluated the copy number of the vector's replicon and analyzed its potential impact on gene expression.

Results: A core hypothesis was proposed: the low-copy replication characteristic of the pORI280 vector may lead to uneven expression levels of the sensor protein, thereby causing system signal instability.

Observations and Records: Based on this hypothesis, the optimization direction was clarified — the next step will prioritize optimizing the sensor module rather than the reporter module.

Participants: Design Group, Signal Analysis Group

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**June 14, 2025 – Cycle 3 Phase Summary**

Objective: To summarize the experimental process and new findings.

Results:

Experiment successful: The background signal issue of the new reporter system has been completely resolved.

Existing shortcomings: The system's signal intensity is unstable, and experimental reproducibility is relatively poor.

Observations and Records: The core issue has shifted from the reporter system to the sensor module, indicating that subsequent optimization should focus on the sensor module.

Participants: The entire team

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**Cycle 3 Summary (May 10 – June 14, 2025)**

Verified Achievements: The reporter system issue has been completely resolved (no background signal detected in the control group throughout the experiment).

Newly Identified Bottleneck: The sensor plasmid exhibits unstable expression, resulting in significant fluctuations in the system's output signal.

Next Steps (Cycle 4): Optimize the PAZ2-1 vector by screening and adopting a vector backbone with a stable copy number.

Timeframe: June 15 – July 11, 2025

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**June 15, 2025 – Problem Localization and Optimization Direction Confirmation**

Objective: To identify a new optimization direction for the system signal instability issue observed in Cycle 3.

Methods and Procedures:

Convened a team meeting to review experimental data and confirm that the root cause of the problem was the unstable expression of PAZ2-1.

Conducted a literature review, confirming that the pORI280 vector used in PAZ2-1 is a low-copy-number vector, whose characteristics tend to cause fluctuations in expression levels.

Results: Through analysis and discussion, it was determined that the core optimization direction would be to replace the vector backbone of PAZ2-1.

Observations and Records: The focus of subsequent work has shifted from "repairing the existing vector" to "screening for new vectors with superior stability."

Participants: Design Group, Signal Analysis Group

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**June 18, 2025 – New Vector Selection**

Objective: To screen and identify a stable vector suitable for PAZ2-1.

Methods and Procedures:

Conducted an extensive investigation into the characteristics (copy number, host range, stability, etc.) of various commonly used plasmid backbones.

After comprehensive evaluation, the pBBR1MCS-5 vector was selected, which offers the key advantages of medium copy number, broad host range, and excellent expression stability.

Results: It was officially confirmed that pBBR1MCS-5 would be used as the new vector backbone for PAZ2-1.

Observations and Records: Based on the characteristics of this vector, it is expected to significantly improve the stability of sensor protein expression and fundamentally alleviate the signal fluctuation issue.

Participants: Literature Research Group

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**June 21, 2025 – PqsR Module Reconstruction**

Objective: To re-clone the PqsR module into the new pBBR1MCS-5 vector.

Methods and Procedures:

Specifically amplified the PqsR gene fragment using PCR technology.

Ligated the amplified PqsR fragment with the pBBR1MCS-5 vector.

Verified the correctness of the cloned product using molecular biology methods (such as sequencing and restriction enzyme digestion).

Results: Successfully constructed the recombinant plasmid pBBR1MCS5-PqsR.

Observations and Records: Verification confirmed that the cloned product sequence was correct and the vector construction was error-free, making it suitable for subsequent experiments.

Participants: Molecular Cloning Group

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**June 23, 2025 – SoxR Module Reconstruction**

Objective: To construct the SoxR module in the pBBR1MCS-5 vector.

Methods and Procedures:

Amplified the SoxR gene fragment using PCR technology.

Inserted the amplified SoxR gene into the pBBR1MCS-5 vector.

Results: Successfully constructed the recombinant plasmid pBBR1MCS5-SoxR.

Observations and Records: The cloning experiment achieved high efficiency, with no obvious ligation failure or fragment loss observed.

Participants: Molecular Cloning Group

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**June 25, 2025 – BrlR Module Reconstruction**

Objective: To construct the BrlR module in the pBBR1MCS-5 vector.

Methods and Procedures:

Specifically amplified the BrlR gene.

Cloned the amplified BrlR gene into the pBBR1MCS-5 vector.

Results: Successfully obtained the recombinant plasmid pBBR1MCS5-BrlR.

Observations and Records: DNA sequencing confirmed that the BrlR gene contained no base mutations, and the constructed plasmid showed good stability during cultivation.

Participants: Molecular Cloning Group

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**June 28, 2025 – MexL Module Reconstruction**

Objective: To construct the MexL module in the pBBR1MCS-5 vector.

Methods and Procedures:

Amplified the MexL gene using PCR technology.

Ligated the MexL gene fragment into the pBBR1MCS-5 vector.

Results: Successfully constructed the recombinant plasmid pBBR1MCS5-MexL.

Observations and Records: The construction experiment achieved a high success rate, and the recombinant plasmid was confirmed to be correct through verification methods such as restriction enzyme digestion and sequencing.

Participants: Molecular Cloning Group

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### July

**July 1, 2025 – System Assembly**

Objective: To co-assemble the newly constructed PAZ2-1 and pMP220 reporter plasmids to form a complete detection system.

Methods and Procedures: The four new PAZ2-1 modules (PqsR, SoxR, BrlR, and MexL) were each co-transformed with their corresponding reporter modules into the chassis strain EC1000Δ*gus*.

Results: All target test strains and supporting control strains were successfully obtained.

Observations and Records: The strain assembly process proceeded smoothly with high overall efficiency and no significant construction failures.

Participants: Chassis Engineering Group

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**July 4, 2025 – Functional Test (Preliminary Round)**

Objective: To determine whether the new system has resolved the signal instability issue.

Methods and Procedures:

Control group: No signaling molecules were added to the strain system.

Experimental group: PQS or PYO signaling molecules were added to induce system response.

Results:

The PqsR, SoxR, and BrlR modules exhibited stable signals, with clear and distinguishable target signals.

The signal intensity of the MexL module in the experimental group was lower than that in the control group.

Observations and Records: All modules except MexL successfully resolved the signal instability issue and functioned as expected.

Participants: Functional Validation Group

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**July 7, 2025 – Multiple Repeat Experiments**

Objective: To verify the reproducibility of the new system.

Methods and Procedures:

Conducted repeated detection of the new system in multiple parallel 96-well plates.

Continuously observed and recorded changes in color/fluorescence intensity for each module.

Results:

The PqsR, SoxR, and BrlR modules maintained consistent signal intensities across multiple plates, showing excellent detection reproducibility.

The MexL module still exhibited relatively weak signals in repeated detections.

Observations and Records: It was confirmed that the overall stability of the new system has been resolved, with only the MexL module currently showing abnormal signal output, which requires further optimization.

Participants: Functional Validation Group

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**July 11, 2025 – Cycle 4 Phase Summary**

Objective: To summarize the results of the current round of PAZ2-1 optimization and system construction.

Results:

Optimization successful: The system signal instability issue has been completely resolved, with stable and reproducible output signals achieved.

Existing problem: The performance of the MexL module has not met expectations, showing weaker signal output.

Observations and Records: The core technical bottleneck (signal instability) has been overcome, marking the project's transition from the "functional debugging phase" to a new stage of "functional realization → application optimization."

Participants: The entire team

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**Cycle 4 Summary (June 15 – July 11, 2025)**

After optimizing the PAZ2-1 vector to pBBR1MCS-5, the system's signal stability was significantly improved.

The performance of the PqsR, SoxR, and BrlR modules fully met the experimental expectations.

The MexL module showed weaker signal output and requires further improvement.

The core functionality of the project has been achieved; the next step will focus on application optimization.

Timeframe: July 12 – Au*gus*t 15, 2025

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**July 12, 2025 – Design Meeting: Entering the Application Optimization Phase**

Objective: On the basis of achieving core functionality, carry out application optimization to enhance the system's safety and practicality.

Methods and Procedures:

Convened a team meeting to focus on discussing the system's biosafety and potential application scenario requirements.

Based on the discussion results, decided to abandon assembling existing vectors and instead design an entirely new dual-plasmid backbone.

Results: Determined the dual-plasmid backbone design strategy, planning to construct pAZ2 (for PAZ2-1) and pBZ2 (for PBZ2-1).

Observations and Records: The project's research and development approach has shifted from "assembling existing vectors" to "independently designing customized vectors," making it more aligned with actual application needs.

Participants: Design Group, HP Group

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**July 15, 2025 – *fabV* Selection Marker Determination**

Objective: To screen and determine an alternative selection marker for the dual-plasmid system, reducing the application risks associated with conventional antibiotic selection.

Methods and Procedures:

Through literature research, suitable safe selection markers for the system were screened, ultimately selecting the *Pseudomonas aeruginosa* endogenous triclosan resistance gene *fabV*.

Confirmed *fabV* as the sole selection marker for the dual-plasmid system to avoid the complexity caused by multiple markers.

Results: Successfully established the specific application plan for the *fabV* gene, including its insertion positions in the dual plasmids and the screening conditions.

Observations and Records: Using triclosan as the screening agent, compared with conventional antibiotics, can effectively reduce false-positive clones and interference from contaminating microorganisms, improving screening accuracy.

Participants: Design Group, Literature Research Group

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**July 18, 2025 – *fabV* Functional Validation**

Objective: To verify whether the *fabV* gene can confer triclosan resistance to the chassis strain *Escherichia coli* (*E. coli*).

Methods and Procedures:

Cloned the *fabV* gene into a vector construct and linked it with the constitutive promoter Pcat to ensure continuous gene expression.

Introduced the construct into *E. coli* and cultured the strain in medium containing triclosan, observing growth status.

Results: *E. coli* containing the *fabV* gene grew normally in triclosan-containing medium, demonstrating that *fabV* successfully conferred triclosan resistance.

Observations and Records: The core functionality of the *fabV* gene has been validated, confirming it meets the basic requirements for use as a selection marker in the dual-plasmid system and can be used in subsequent system construction.

Participants: Molecular Cloning Group

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**July 22, 2025 – Inducible Control Validation of *fabV***

Objective: To verify whether the *fabV* gene can achieve strictly inducible expression through the pBAD promoter, enhancing system safety.

Methods and Procedures:

Inserted the *fabV* gene downstream of the pBAD promoter to construct an inducible expression vector.

Cultured *E. coli* containing this vector in media with and without arabinose (inducer), observing the resistance phenotype.

Results:

With arabinose: Cells grew normally, showing triclosan resistance.

Without arabinose: Cells failed to grow, losing triclosan resistance.

Observations and Records: The experiment demonstrated that *fabV* can achieve strictly controllable expression through the pBAD promoter — "expression upon induction, silencing without induction." This can serve as a "safety lock" for the dual-plasmid system, reducing biosafety risks.

Participants: Molecular Cloning Group

---

**July 27, 2025 – pAZ2 Backbone Assembly**

Objective: To construct the new PAZ2-1 backbone plasmid pAZ2.

Methods and Procedures:

Selected the pORI280 replicon (ori), leveraging its host range restriction characteristics to enhance system safety.

Integrated the *fabV*-pBAD inducible expression module into the vector, followed by the T1T2 terminator to ensure precise regulation of gene expression.

Results: Successfully constructed the PAZ2-1 backbone plasmid pAZ2 with the structure: ori-araC-pBAD-*fabV*-T1T2.

Observations and Records: pAZ2 implements a dual safety lock through "pORI280 ori host restriction" and "*fabV*-pBAD controllable expression," significantly enhancing biosafety.

Participants: Vector Engineering Group

---

 **July 30, 2025 – pBZ2 Backbone Assembly**

Objective: To construct the new PBZ2-1 backbone plasmid pBZ2.

Methods and Procedures:

Adopted the pBluescript replicon (ori, ColE1 type) to ensure compatibility with the pORI280 ori of pAZ2.

Designed the *fabV* gene to be driven by the constitutive promoter Pcat, ensuring continuous expression of the selection marker.

Results: Successfully obtained the PBZ2-1 backbone plasmid pBZ2 with the structure: ori-Pcat-rbs-*fabV*-T1T2.

Observations and Records: The replicon types of pBZ2 and pAZ2 are compatible, ensuring stable coexistence of the two plasmids in the same host strain, which lays the foundation for the dual-plasmid system.

Participants: Vector Engineering Group

---

### August

**August 3, 2025 – System Reconstruction**

Objective: To integrate the validated sensor modules and reporter modules into the new backbones, constructing a complete dual-plasmid system.

Methods and Procedures:

Transferred the four sensor modules into pAZ2.

Transferred the four reporter modules into pBZ2.

Results: Successfully obtained the complete new dual-plasmid system (pAZ2-sensor modules + pBZ2-reporter modules).

Observations and Records: The integration of modules with the new backbones proceeded smoothly, with no abnormal fragment ligation or plasmid construction failures. The system assembly efficiency met expectations.

Participants: Chassis Engineering Group

---

**August 7, 2025 – Functional Regression Test**

Objective: To verify whether the sensor-reporter system integrated into the new backbone (pAZ2/pBZ2) maintains its original functionality.

Methods and Procedures: Repeated the Cycle 4 induction experiment protocol:

Control group: No signaling molecules added to the strain system.

Experimental group: PQS or PYO signaling molecules added to the strain system.

Results:

Control group: No background signal detected; background interference was zero.

Experimental group: Clear, stable, and reproducible target signals were successfully generated.

Observations and Records: The system performance on the new backbone was highly consistent with the results from Cycle 4, confirming that module integration did not affect core functionality.

Participants: Functional Validation Group

---

**August 12, 2025 – Repeat Validation and Stability Assessment**

Objective: To further verify the long-term stability and batch consistency of the new dual-plasmid system.

Methods and Procedures: System functionality tests were repeatedly conducted under conditions of different medium batches and various culture durations.

Results: In all batches of experiments, the signal intensity output by the system remained consistent without significant fluctuations, showing excellent reproducibility.

Observations and Records: The new system demonstrated stable performance across multiple culture batches, fully meeting the stability requirements for practical application.

Participants: Functional Validation Group

---

**August 15, 2025 – Cycle 5 Phase Summary**

Objective: To summarize the experimental progress and key achievements of Cycle 5.

Results:

Successfully constructed the new dual-plasmid backbones pAZ2 and pBZ2.

Completed functional validation of the *fabV* gene, realizing a strictly controllable and safe selection system.

The complete sensor-reporter system functions normally on the new backbones, with stable and reliable signals.

Observations and Records: The new system has advanced from the "basic functionality realization" stage to a mature stage characterized by "high safety + high application potential."

Participants: The entire team.

---

**Cycle 5 Summary (July 12 – Au*gus*t 15, 2025)**

Independently constructed new plasmid backbones, significantly enhancing system safety and practical applicability.

Introduced the *fabV* triclosan resistance system, improving screening specificity and greatly reducing false-positive clones.

Successfully achieved the dual goals of "maintaining core functionality + enhancing biosafety," laying a solid foundation for application optimization in Cycle 6.

Timeframe: Au*gus*t 16 – September 19, 2025

---

**August 16, 2025 – Multi-module Sensor Design Meeting**

Objective: To optimize the system for improved detection sensitivity and specificity based on single-module sensing.

Methods and Procedures:

Reviewed previous experimental data and analyzed optimization directions in combination with modeling predictions.

Decided to construct a pqsR + soxR dual-sensor system.

Designed the reporter module to carry both PpqsA-*lacZ* and PmexG-*gus* response elements.

Results: The design plan for the dual-sensor-dual-reporter composite system was confirmed, entering the construction preparation phase.

Observations and Records: In previous tests, the soxR-PmexG module exhibited the strongest signal intensity and will serve as the key functional module of the composite system.

Participants: Design Group, Modeling Group

---

**August 20, 2025 – PAZ2-1 Composite Construction**

Objective: To achieve simultaneous expression of the pqsR and soxR genes on the pAZ2 backbone.

Methods and Procedures:

Specifically amplified the pqsR and soxR genes by PCR.

Adopted a dual-promoter expression strategy to construct the expression unit:

Pcat-RBS-pqsR-T0T1 + Pcat-RBS-soxR-T0T1, and inserted it into the pAZ2 backbone.

Results: Successfully constructed the composite PAZ2-1 plasmid (pAZ2-pqsR+soxR).

Observations and Records: Both genes achieved stable expression on the same backbone. The multi-gene co-expression construct was successful without expression conflicts.

Participants: Molecular Cloning Group

---

**August 23, 2025 – PBZ2-1 Composite Construction**

Objective: To simultaneously carry dual reporter modules on the pBZ2 backbone.

Methods and Procedures:

Inserted the two response elements PpqsA-*lacZ* and PmexG-*gus* into the pBZ2 backbone.

Verified the assembly order and correctness of the two modules through sequencing and restriction enzyme digestion.

Results: Successfully obtained the composite PBZ2-1 plasmid (pBZ2-PpqsA-*lacZ*+PmexG-*gus*).

Observations and Records: The dual reporter modules were correctly assembled on the pBZ2 backbone, with no fragment deletions or orientation errors. The construction task was completed smoothly.

Participants: Molecular Cloning Group

---

**August 25, 2025 – Dual-Plasmid Composite System Assembly**

Objective: To co-transform PAZ2-1 and composite PBZ2-1 into the chassis strain EC1000Δ*gus*.

Methods and Procedures:

Used electroporation to simultaneously introduce composite PAZ2-1 and composite PBZ2-1 into EC1000Δ*gus*.

Screened positive clones using selection markers.

Results: Successfully obtained the composite sensor-reporter strain.

Observations and Records: Positive clones showed normal colony growth without growth retardation or morphological abnormalities. The strain can be used for subsequent functional testing.

Participants: Chassis Engineering Group

---

**August 28, 2025 – Medium Comparison Experiment**

Objective: To compare the effects of different media on pyocyanin (PYO) production by *Pseudomonas aeruginosa* and determine the optimal culture medium.

Methods and Procedures:

Cultured *P. aeruginosa* in both LB medium and PB medium.

Measured PYO (green pigment) production in both media and determined β-galactosidase activity.

Results: PB medium showed significantly better performance than LB medium. The PB group exhibited higher PYO production and correspondingly stronger detection signals.

Observations and Records: PB medium effectively promotes PYO synthesis and was determined to be the standard medium for subsequent experiments.

Participants: Functional Validation Group

---

### September

**September 1, 2025 – Composite Culture System Setup**

Objective: To optimize the detection system by adding enzyme substrates to PB medium, thereby improving detection efficiency.

Methods and Procedures:

Mixed PB medium with MMO-MUG (enzyme substrate) to prepare PB+MMO-MUG composite medium.

Verified whether this medium could support the growth of both *E. coli* and *P. aeruginosa*.

Results: The PB+MMO-MUG composite medium system operated successfully without growth inhibition.

Observations and Records: This composite medium enabled simultaneous detection under dual-bacteria co-culture conditions, eliminating the need for separate cultures and significantly improving detection efficiency.

Participants: Culture Optimization Group

---

**September 5, 2025 – Small-Volume Culture Exploration**

Objective: To optimize the detection system for small-volume environments, laying the foundation for subsequent high-throughput detection.

Methods and Procedures:

Cultured *Pseudomonas aeruginosa* in 1.5 mL centrifuge tubes and 96-well plates (small-volume systems).

Detected PYO synthesis in the small-volume systems.

Results: Under small-volume culture conditions, bacterial growth was significantly limited, and PYO production was extremely low, failing to meet detection requirements.

Observations and Records: The main reason for insufficient PYO synthesis in small-volume cultures was insufficient dissolved oxygen, which restricted bacterial metabolic activity.

Participants: Culture Optimization Group

---

**September 8, 2025 – Oxygen Supply Method Exploration**

Objective: To address the issue of insufficient dissolved oxygen in small-volume culture systems and restore normal PYO synthesis.

Methods and Procedures: Tested multiple oxygen supply methods:

1. Mechanical oxygenation using a shaker.

2. Chemical oxygen donors (NaHCO₃, H₂O₂, Na₂CO₃·1.5H₂O₂, KNO₃).

3. External gas-permeable membranes (EPTFE membrane and nanoscale gas-permeable membrane).

Results:

Mechanical oxygenation using a shaker showed limited effectiveness.

Chemical oxygen donors had toxic side effects (inhibiting bacterial growth).

Adding an EPTFE membrane significantly improved the oxygenation environment and successfully restored PYO synthesis.

Observations and Records: The EPTFE gas-permeable membrane was identified as the optimal solution for addressing oxygenation issues in small-volume systems, showing no side effects and providing stable performance.

Participants: Culture Optimization Group

---

**September 12, 2025 – High-Throughput Detection Validation**

Objective: To verify the feasibility of applying the composite sensor-reporter system in a 96-well plate (small-volume, high-throughput scenario).

Methods and Procedures:

Operated the composite system in the optimized medium system: PB + MMO-MUG + triclosan + EPTFE gas-permeable membrane.

Added *Pseudomonas aeruginosa* and detected the system's fluorescent signal and color reaction.

Results: The composite system stably detected the target bacterium's signal in the small-volume 96-well plate format, with no significant fluctuations.

Observations and Records: The small-volume, high-throughput detection protocol was successfully validated, laying a core foundation for subsequent real environmental sample testing.

Participants: Functional Validation Group

---

**September 16, 2025 – Results Analysis Meeting**

Objective: To summarize the experimental results of Cycle 6 and analyze the optimization effectiveness and system performance improvements.

Methods and Procedures:

Compared the effects of different culture systems (LB / PB / PB+MMO-MUG) and different oxygen supply methods (shaker / chemical oxygen donors / EPTFE membrane).

Collected and discussed the signal intensity, sensitivity, and specificity performance of the composite sensor in the optimized system.

Results:

The dual-sensor modules (pqsR + soxR) acted synergistically, significantly improving the system's detection sensitivity and specificity for the target bacterium.

The "PB + MMO-MUG + EPTFE gas-permeable membrane" system provided a stable and efficient detection environment, ensuring signal reliability.

Observations and Records: After multi-dimensional optimization, the practical application potential of the composite system has been greatly enhanced, fully meeting the core requirements for high-throughput detection.

Participants: Design Group, Functional Group, HP Group

---

**September 19, 2025 – Cycle 6 Phase Summary**

Objective: To conclude Cycle 6 and clarify the phase achievements and next-stage plans.

Results:

Successfully constructed and validated the pqsR + soxR dual-sensor composite system, with core performance meeting the targets.

Established a high-throughput detection platform suitable for 96-well plates, solving the key technical challenges of small-volume culture.

Observations and Records: Cycle 6 has achieved the "laboratory optimization" goal. The next stage (Cycle 7) will move to real water sample testing to verify the system's applicability in actual environmental conditions.

Participants: The entire team.

---

**Cycle 6 Summary (Au*gus*t 16 – September 19, 2025)**

1.Constructed and validated a multi-module sensing combination (pqsR + soxR), improving detection performance.

2.Optimized the culture system: PB medium outperformed LB; PB + MMO-MUG enabled dual-bacteria co-culture.

3.Resolved the small-volume oxygenation challenge: EPTFE gas-permeable membrane proved to be the optimal solution.

4.Established a stable high-throughput detection system, paving the way for environmental sample testing.

Timeframe: September 20 – October 1, 2025

---

**September 20, 2025 – Final System Confirmation Meeting**

Objective: To finalize the detection system for the ultimate application validation.

Methods and Procedures:

Confirmed the sensor module: pqsR + soxR dual-sensor.

Confirmed the reporter module: PpqsA-*lacZ* + PmexG-*gus* dual-reporter.

Confirmed the culture system: PB + MMO-MUG medium + triclosan selection pressure.

Confirmed the oxygen supply condition: 96-well plate + EPTFE gas-permeable membrane.

Results: The final detection system was confirmed.

Observations and Records: All optimization achievements have been integrated into the final system.

Participants: The entire team.

---

**September 22, 2025 – Environmental Water Sample Collection**

Objective: To collect and process various real water samples in preparation for actual detection experiments.

Methods and Procedures:

Collected four types of typical water samples: lake water, tap water, domestic sewage, and purified water.

Performed sterile treatment on all samples to avoid contamination by miscellaneous bacteria, and assigned unique identification numbers.

Results: Successfully obtained four types of environmental water samples that meet the experimental requirements.

Observations and Records: The entire water sample processing was carried out in a sterile environment, with special attention to preventing secondary contamination from container pollution and operational contamination, ensuring sample authenticity.

Participants: Sample Collection Group

---

**September 24, 2025 – Sample Pretreatment**

Objective: To pretreat the collected environmental water samples to ensure their suitability for the final detection system.

Methods and Procedures:

Removed large particulate impurities from the water samples through filtration to prevent clogging of detection wells or interference with bacterial growth.

Measured and adjusted the pH value and salinity of the water samples to a range suitable for the growth of *E. coli* and *P. aeruginosa*.

Results: All water samples were successfully pretreated, with key physical and chemical indicators meeting the required standards.

Observations and Records: The properties of the pretreated water samples are close to standard experimental conditions, and will not interfere with detection results due to environmental factors. These samples are ready for subsequent detection using the engineered strains.

Participants: Sample Processing Group

---

**September 26, 2025 – Engineered Bacteria Detection Experiment**

Objective: To run the final engineered bacteria detection system on pretreated real water samples and verify its practical application effectiveness.

Methods and Procedures:

Added the four types of pretreated water samples into a 96-well plate.

Inoculated each well with the composite engineered bacteria, while setting up a blank control group without the target bacteria.

Cultured under optimized conditions, and observed/recorded the color changes and fluorescent signal intensity in each well.

Results:

Lake water and domestic sewage showed obvious coloration/fluorescence.

Tap water and purified water showed no signal.

Observations and Records: The actual water sample detection results fully matched expectations (higher probability of detecting target bacteria in lake water and domestic sewage), confirming that the final detection system has the capability for real environmental applications.

Participants: Functional Validation Group

---

**September 28, 2025 – GB Standard Method Parallel Detection**

Objective: To compare the detection results of the engineered bacteria detection system with the national standard method (GB method) and verify the accuracy of the engineered bacteria system.

Methods and Procedures:

Used the GB method to perform parallel detection on the same batch of four water samples (lake water, tap water, domestic sewage, purified water).

Recorded the GB method detection results and compared them one by one with the previous engineered bacteria detection results.

Results:

The positive/negative determination results of the two detection methods were completely consistent.

The engineered bacteria detection system showed shorter color reaction time, required no complex instrument operation, and had a simpler workflow.

Observations and Records: The experiment directly proved the accuracy and practicality of the engineered bacteria detection system, whose performance is not lower than the national standard method, and which has an advantage in efficiency.

Participants: Control Experiment Group

---

**September 30, 2025 – System Stability Verification**

Objective: To confirm the stability and repeatability of the final engineered bacteria detection system across different experimental batches.

Methods and Procedures:

Repeatedly conducted detection experiments on lake water and domestic sewage (positive water samples), performing multiple parallel tests across batches.

Compared key indicators such as signal intensity and color development time among different batches.

Results: The detection results were completely consistent across batches. The target signal intensity remained stable without fluctuation, and the differences in reaction time were within the acceptable range.

Observations and Records: The final system demonstrates excellent batch-to-batch repeatability, requiring no frequent parameter adjustments and meeting the stability requirements for practical applications.

Participants: Functional Validation Group

---

### October

**October 1, 2025 – Cycle 7 Phase Summary**

Objective: To summarize the application validation results of the final engineered bacteria detection system and clarify its value and future directions.

Results:

The detection results of the engineered bacteria system were highly consistent with the national standard GB method, meeting accuracy requirements.

The engineered bacteria system exhibited faster reaction speed, simpler operation, and significantly improved detection efficiency.

The system has demonstrated core potential for application in environmental water monitoring.

Observations and Records: The project has officially moved from the "laboratory optimization" stage to the "future feasibility exploration" stage. Future directions include:

Expanding detection to more scenarios such as industrial wastewater and medical sewage

Exploring integration with portable devices to enable on-site rapid detection

Participants: The entire team.

---

**Cycle 7 Summary (September 20 – October 1, 2025)**

System Integration Complete: Dual-sensor + dual-reporter + optimized culture system + high-throughput detection environment.

Real Water Sample Validation:

Lake water / domestic sewage → positive

Tap water / purified water → negative

Consistent with Standards: Results fully aligned with the national standard GB method.

Advantages: Fast detection speed, simple operation, strong scalability, and potential for on-site testing.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block header %}

![示例图片](https://static.igem.wiki/teams/5853/image/otherimage/project.webp)

{% endblock %}
{% block page\_content %}

**### Cycle 1: Chassis Construction and Preliminary Validation of the Sensing Module**

---

**Design**

The core of our project is to construct an engineered bacterium capable of detecting the quorum sensing signal molecules (PQS, PYO) of *P. aeruginosa*. To this end, our first engineering cycle set two main design goals:

1.Construct a "Clean" Chassis Cell

Selected Chassis: We chose E. coli EC1000 as the chassis because it can support the pORI280 replicon plasmid we planned to use.

Background Elimination: We planned to use *lacZ* and *gusA* as reporter genes. Through literature review and experimental verification, we found that although EC1000 is *lacZ* deficient, it possesses a functional *gusA* gene on its genome. To avoid background interference, we decided it was necessary to knock out the endogenous *gusA* gene, providing a "zero-background" platform for the subsequent GUS reporter system.

2.Design a Modular Sensing-Reporter System

We designed a two-plasmid system to achieve flexible modular testing:

Plasmid A (Sensing Module): Carries a constitutively expressed promoter (Pcat) driving the sensor protein (e.g., PqsR, MexL, SoxR, BrlR).

Plasmid B (Reporter Module): Carries a response promoter corresponding to the sensor protein (e.g., PpqsA, PphzA1, PmexG, PbrlR), used to drive the reporter gene (*lacZ* or *gusA*).

We planned to test four different sensing-reporter module pairs in parallel to screen for the best-performing combination.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e1.webp)

Figure 1. Plasmid A (Sensor Module): Carries a constitutively expressed promoter (Pcat) driving the sensor protein (e.g., PqsR, MexL, SoxR, BrlR)

![示例图片](https://static.igem.wiki/teams/5853/cycle/e2.webp)

Figure 2. Plasmid B (Reporter Module): Carrying a responsive promoter corresponding to the sensor protein (e.g., PpqsA, PphzA1, PmexG, PbrlR), and using it to drive the reporter gene (*lacZ* or *gusA*).

---

**Build：**

Following our design, we completed a large amount of recombinant vector construction work:

1.Successfully constructed the EC1000Δ*gusA* mutant strain:

We obtained the homologous recombination fragment for *gusA* using Overlap Extension PCR. Utilizing the pDM4 suicide plasmid system, through conjugation transfer, single-crossover screening, and sucrose counter-selection, we successfully and seamlessly knocked out the *gusA* gene on the EC1000 genome. Through PCR and phenotypic verification, we finally confirmed the acquisition of the genetically clean chassis bacterium E. coli EC1000Δ*gusA*.

2.Completed the construction of the four sets of sensing-reporter plasmids:

We successfully constructed all four Plasmid A (sensing modules) based on plasmids pORI280, pDM4, pET28b, pUC18T-mini-TN7T, and p34SGm, and added a gentamicin resistance gene (Gm) to each module for selection: Ori -Pcat-RBS-pqsR-T0T1-Gm; Ori -Pcat-RBS-soxR-T0T1-Gm; Ori -Pcat-RBS-brlR-T0T1-Gm; Ori -Pcat-RBS-mexL-T0T1-Gm.

We also constructed the four Plasmid B (reporter modules). Among them, the first set used a *lacZ* reporter system based on the mini-CTX vector, while the other three sets cloned the *gusA* gene amplified from EC1000 into the pUC18T-mini-TN7T-Gm vector.

Finally, we co-transformed the corresponding sensing and reporter plasmids for each module into the EC1000Δ*gusA* chassis, obtaining all test strains and their corresponding negative control strains.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e3.webp)

Figure 3. Fluorescence phenotypic verification of EC1000 Δ*gusA* gene knockout.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e4.webp)

Figure 4. Recombinant Expression Plasmid A: Structure and Element Origins

---

**Test**

We performed functional tests on the constructed strains. The experimental groups were treated with their corresponding signal molecules (PQS or PYO), and the control groups (CK, reporter plasmid without the response promoter) were treated similarly.

Expected Result: In the experimental group with the MexL-PphzA1 element set, the fluorescence intensity was significantly lower compared to that of the control group. For the other three groups, only the experimental groups produced color (yellow substance or blue fluorescence) due to the expression of the reporter gene after the addition of the signal molecule; under no circumstances should the control groups show any color change.

Actual Result: The results were completely beyond our expectations. We observed that there was almost no difference in fluorescence intensity between the experimental group and the control group in the MexL-PphzA1 element set, and the control groups of all three sets produced obvious color signals.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e5.webp)

Figure 5. PqsR-PpqsA element test; B: SoxR-PmexG element test; C: BrlR-PbrlR element test; D: MexL-PphzA1 element test.

---

**Learn and Conclude**

Although this result was a failure, it provided extremely valuable learning outcomes. The abnormal signal from the control groups indicated a serious problem of background interference in our system. To identify the root cause, we conducted a series of troubleshooting steps:

1.Investigating the Promoter Elements: We first suspected the response promoters (PpqsA, etc.) we used. Therefore, we cloned them into their native host, *P. aeruginosa* PAO1, for testing. The results showed that these promoters functioned completely normally in PAO1, with very low background and high inducible activity (Figure 3A). This proved that our promoter elements themselves were not the problem.

2.Investigating the Reporter Vectors: Since the promoters were fine, the problem must lie in the backbone of the reporter plasmids. For the *lacZ* system: We transformed the mini-CTX-*lacZ* plasmid containing no promoter into E. coli TG1 and found that this strain produced very high β-galactosidase activity (Figure 6B). This confirmed that the mini-CTX-*lacZ* vector itself has extremely high constitutive expression in E. coli. The mini-CTX vector is designed as an integrative vector for *P. aeruginosa*. It stably integrates the gene of interest as a single copy into the attB site of the *P. aeruginosa* chromosome via site-specific recombination. This 'single-copy integration' mode ensures very low background expression, which was our initial reason for choosing it. However, it is not designed for E. coli. The E. coli genome lacks the attB integration site. Therefore, mini-CTX-*lacZ* cannot integrate and instead relies on its own E. coli replication origin (pUC ori), replicating as a high-copy plasmid, making it unsuitable as a low-background reporter vector.

3.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e6.webp)

Figure 6. A:Enzyme activity validation of four promoters (pqsA, phzA1, mexG, brlR) in *P. aeruginosa* PAO1 B: 2. Investigating the Reporter Vectors (mini-CTX-*lacZ*) in E. coli TG1.

For the gus system: For the other three groups, the control used the pUC18T-mini-TN7T-gus-Gm plasmid without the response promoter. These also produced fluorescence. We speculated that the inherent Plac promoter upstream in the pUC vector backbone was directly driving the expression of the downstream gus gene.

---

**Conclusion of this cycle:**

Although we successfully completed complex chassis modification and plasmid construction, the entire system failed functionally due to the erroneous selection of the reporter plasmid vector backbones. The most important lesson we learned is: Before constructing a complex system, it is essential to rigorously and independently characterize the performance (especially the background leakage level) of every basic component (especially the vector) in the target chassis.

---

**Next Step / Redesign:**

Based on the profound lesson from this cycle, the goal of our next cycle is very clear: Abandon the currently used *lacZ* and gus reporter plasmid vectors, re-screen for a reliable, low-background reporter vector, and then re-test our sensing modules in combination with it.

---

**### Cycle 2: Re-exploration after Changing the Reporter Vector**

---

**Redesign:**

Based on the conclusion of the first cycle—our failure stemmed from choosing unsuitable reporter plasmid vectors—the design goal for our second cycle was very clear: switch to a completely new, theoretically more reliable reporter vector backbone.

New Choice: We selected the widely used expression vector pET28b as our new "Plasmid B" platform. Our hypothesis was that this classic vector would provide a low-background expression environment for our reporter genes (*lacZ* and gus).

Keeping Variables Constant: To accurately verify the performance of the new vector, we kept all other successful components unchanged:

Chassis Cell: Still used our carefully constructed EC1000Δ*gusA*.

Plasmid A: Remained unchanged (ori-Pcat-rbs-pqsR/mexL/brlR/soxR-T0T1-Gm).

Our core idea was to fix the entire system by replacing the failed variable (Plasmid B).

---

**Build**

We quickly executed the new construction plan:

1.Constructed new reporter plasmids: We cloned the four response promoter-reporter gene elements (PpqsA-*lacZ*, PphzA1-gus, PbrlR-gus, PmexG-gus) into the pET28b vector respectively.

2.Constructed new control groups: Simultaneously, we constructed pET28b control plasmids without the response promoter (pET28b-*lacZ* and pET28b-gus).

3.Assembled test strains: We co-transformed the new reporter plasmids with the original sensing plasmids into EC1000Δgus, successfully obtaining all newly designed test strains and control strains.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e7.webp)

Figure 7. Schematic diagram of skeleton construction of new plasmid B in this cycle.

---

**Test**

We conducted functional tests on all new strains under exactly the same experimental conditions as the first cycle and observed the results.

Expected Result: In the experimental group with the MexL-PphzA1 element set, the fluorescence intensity was significantly lower compared to that of the control group. For the other three groups, only the experimental groups produced color (yellow substance or blue fluorescence) due to the expression of the reporter gene after the addition of the signal molecule; under no circumstances should the control groups show any color change.

Actual Result: Contrary to our expectations, we observed the exact same failure pattern as in the first cycle.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e8.webp)

Figure 8. A: PqsR-PpqsA element test; B: SoxR-PmexG element test; C: BrlR-PbrlR element test; D: MexL-PphzA1 element test.

---

**Learn and Conclude**

Two consecutive failures forced us to reflect more deeply. The problem was clearly more complex than just "choosing the wrong vector." This failure led us to shift the focus of our analysis from "which vector" to "what element on the vector is causing the problem."

New Hypothesis: We carefully analyzed the pET28b vector map and proposed a new hypothesis: the problem might lie with the vector's inherent kanamycin resistance gene (KanR). We speculated that the promoter driving KanR expression might cause "transcriptional read-through," accidentally initiating the expression of our inserted, supposedly silent reporter gene, thus causing our failure.

---

**Conclusion of this cycle:**

The second failure taught us that we must pay attention to any element on the vector (especially resistance genes) that might cause unintended "crosstalk" with our inserted genetic circuit. Simply replacing the vector without considering the internal interactions of its components is far from sufficient.

---

**Next Step / Redesign**

This time, our redesign will be more targeted, aiming to thoroughly solve the "crosstalk problem":

We decided to change the "Plasmid B" vector again. But this time, our selection criteria will be stricter. We are not just looking for a new vector, but for one designed to minimize interference from internal components. For example, a vector where the resistance gene is transcribed in the opposite direction to our insert. If we cannot find one, we will modify one ourselves, for instance, by flanking our reporter gene element with strong bidirectional terminators to actively "shield" it from any irrelevant transcription from the vector backbone.

---

**### Cycle 3: Targeted Problem Solving and the Dawn of Success**

---

**Redesign**

Learning from the profound lesson of failure due to "transcriptional read-through" in the previous two cycles, our third redesign became unprecedentedly rigorous and targeted. Our design philosophy shifted from "trying a different vector" to "fundamentally eliminating the possibility of 'crosstalk'."

Carefully Selected "Plasmid B" Vector: We chose the pMP220 vector as our new reporter platform. The reasons for choosing it were very clear and directly addressed our previous issues:

1. No Endogenous Promoter: The multiple cloning site (MCS) region of this vector is very "clean," containing no known promoter elements;

2. Reverse Orientation of Resistance Gene: The transcription direction of its resistance gene is completely opposite to the direction of our insert, physically preventing the possibility of "transcriptional read-through" interference.

Isolating Variables: We still kept the "Plasmid A" sensing plasmid and the EC1000Δgus chassis unchanged to ensure we could clearly evaluate whether the pMP220 vector truly solved the problem.

---

**Build**

According to the new design, we systematically rebuilt our " Plasmid B" reporter plasmid library:

1.Constructed the pMP220 reporter system: We successfully cloned the four response promoter-reporter gene elements into the pMP220 vector and constructed the corresponding negative control plasmids.

2.Assembled the final strains: Co-transformed the updated "Plasmid B" with the original "Plasmid A" into the EC1000Δ*gusA* chassis again, preparing for the final verification.

3.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e9.webp)

Figure 9. The four sets of responsive promoter-reporter gene elements were cloned into the pMP220 vector

---

**Test**

We performed the functional test for the third time.

Expected Result: In the experimental group with the MexL-PphzA1 element set, the fluorescence intensity was significantly lower compared to that of the control group. For the other three groups, only the experimental groups produced color (yellow substance or blue fluorescence) due to the expression of the reporter gene after the addition of the signal molecule; under no circumstances should the control groups show any color change.

Actual Result: The results are almost consistent with the expected results.

New Challenge: For the MexL-PphzA1 element, the fluorescence intensity of the experimental group was slightly lower compared to that of the control group. For the other three sets of elements, in the experimental groups, although we observed induction-dependent signal production, the intensity of the signals was extremely unstable. There were significant differences in the depth of the yellow substance among multiple repeated experiments and different sample wells, lacking good reproducibility.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e10.webp)

Figure 10. A: PqsR-PpqsA element test; B: SoxR-PmexG element test; C: BrlR-PbrlR element test; D: MexL-PphzA1 element test]

---

**Learn and Conclude**

The third cycle was a critical turning point in our project. We finally overcame the problem that had plagued us for so long, proving that the basic design logic of our system was feasible.

However, the new problem of signal instability shifted our attention from the "Plasmid B " (reporter module) to the "Plasmid A " (sensing module).

New Hypothesis: Since the reporter system was now functional, the root cause of the unstable output signal likely lay in the concentration of the sensor proteins (PqsR, MexL, etc.) produced by the "Plasmid A" plasmid. We therefore hypothesized that the expression from our "Plasmid A" plasmid might be unstable. This could be due to the low and uneven copy number of its pORI280 replicon, leading to large fluctuations in the intracellular concentration of the sensor protein across different cells or growth states, thereby making the entire system's response unpredictable.

---

**Conclusion of this cycle:**

We successfully constructed a usable reporter platform. Simultaneously, we identified the next bottleneck in system performance—the instability of the sensing module expression. The main contradiction of the project shifted from "can it work" to "can it work stably."

---

**Next Step / Redesign**

Our engineering modification path entered a new stage. The goal of the next cycle would focus entirely on our "Plasmid A" sensing plasmid:

We will systematically study and optimize the expression stability of the "Plasmid A", replacing it with a vector with medium expression level and medium copy number.

---

**### Cycle 4: Optimizing the Stability of the Sensing Module**

---

**Redesign：**

In the third cycle, we successfully constructed a functional sensing-reporting system but also identified a new bottleneck: unstable sensing signals. Our hypothesis was that the problem stemmed from unstable expression of the "Plasmid A" sensing plasmid (ori-pqsR, etc.). Therefore, the design goal of the fourth cycle focused entirely on improving the stability of the sensing module.

Core Strategy: Abandon the previously used pORI280 replicon and replace the "Plasmid A" with a plasmid backbone with a more stable and uniform copy number.

New Choice: We selected the pBBR1MCS-5 vector. This is a well-known, medium-copy-number, broad-host-range plasmid, renowned for its stable copy number maintenance in various bacterial strains. We predicted that by cloning our sensing elements into pBBR1MCS-5, we could ensure a stable and sufficient concentration of the sensor protein inside the cell, thereby making the entire system's response stable and reproducible.

Maintaining Controls: We continued to use the proven successful "Plasmid B" pMP220 reporter system from the third cycle to ensure we only changed one variable.

---

**Build：**

According to the new design, we comprehensively rebuilt the "Plasmid A" sensing plasmids:

Rebuilt the Sensing Plasmid Library: We re-cloned all four sensing modules (PqsR, MexL, BrlR, SoxR) into the pBBR1MCS-5 vector backbone.

Assembled the Final System: We co-transformed the newly constructed, more stable "Plasmid A" plasmids with our reliable "Plasmid B" reporter plasmids into the EC1000Δgus chassis, obtaining the final version of our test strains.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e11.webp)

Figure 11. All four sensor modules (PqsR, MexL, BrlR, SoxR) were re-cloned into the pBBR1MCS-5 vector backbone, along with two control plasmids.

---

**Test**

We performed induction and detection for all four module sets.

Expected Result: Control groups show no signal, experimental groups produce clear, stable, and reproducible signals after induction.

Actual Result: For the PqsR, BrlR, and SoxR modules, the control groups showed no signal, while the experimental groups produced clear, stable, and reproducible signals after induction. For the MexL module, the fluorescence intensity of the experimental group was significantly lower compared to that of the control group.Clean Background: As in the third cycle, all negative control groups showed no color or fluorescence signal, exhibiting a perfect "off" state.

Clear and Stable Signals: After adding the corresponding signal molecules, the experimental groups of all four sets produced clear, visible signals. Most crucially, in multiple replicate experiments, the signal intensity showed high consistency and reproducibility, completely solving the previously observed signal fluctuation problem.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e12.webp)

Figure 12. A: PqsR-PpqsA element test; B: SoxR-PmexG element test; C: BrlR-PbrlR element test; D: MexL-PphzA1 element test]

---

**Learn and Conclude：**

The fourth engineering cycle marked the complete realization of our project's core functionality. Through a series of systematic troubleshooting and iterative design, we ultimately constructed a high-performance, high signal-to-noise ratio, high-stability two-plasmid biosensor system.

Successful Hypothesis Verification: The success of this experiment strongly verified the hypothesis we proposed in the third cycle—the root cause of the previous signal instability was indeed the unstable expression of the "Plasmid A" sensing plasmid. By switching to the more copy-number-stable pBBR1MCS-5 vector, we provided a stable expression platform for the sensor protein, thereby ensuring the robustness of the entire genetic circuit's response.

---

**Conclusion of this cycle:**

Through this cycle, we solved the core technical challenge of signal instability and obtained a functional biosensor.

---

**Next Step：**

The success of the fourth cycle marked that we had fully mastered the key variables affecting sensor performance and possessed a prototype that performed stably and reliably under laboratory conditions. At this point, the focus of our project began to shift from "functional realization" to "application optimization," i.e., how to enable our engineered bacteria to safely and reliably leave the laboratory in the future.

In this new phase, biosafety and the long-term stability of the two-plasmid system became our two equally important core design goals.

We conducted a comprehensive review and weighing of the existing system:

First, the trade-off between performance and safety: The pBBR1MCS-5 vector, while providing stable signals, poses a biosafety risk due to its broad-host-range characteristic. Conversely, the pORI280 ori, although performing poorly in the previous backbone, possesses a host-dependent characteristic which is a valuable biocontainment feature we are reluctant to abandon.

The stability challenge of the two-plasmid system: For the Plasmid A and Plasmid B plasmids to stably coexist in the same cell for a long time without excluding each other, they must have incompatible replication origins (ori).

Based on the above considerations, we decided to no longer be satisfied with "piecing together" existing vectors, but to design and construct from scratch a set of bran-new, customized two-plasmid backbones integrating high performance, high safety, and high stability. This became the core task of our fifth cycle.

Our redesign will follow these three major strategies:

1.Construct a Dual-Locked "Safe Plasmid A":

We will specially build a new backbone for the sensing plasmid (Plasmid A). This backbone will use the pORI280 ori, achieving host restriction (first safety lock). Simultaneously, we will place the fabV resistance gene on the Plasmid A under the strict control of an arabinose-inducible promoter (pBAD). This means the Plasmid A can only be maintained under selection pressure when arabinose is supplied in the laboratory. This constitutes a controllable "kill switch" (second safety lock).

2.Construct a Compatible and Efficient "Plasmid B":

To solve the plasmid compatibility issue, we will build a new backbone for the reporter plasmid (Plasmid B) using a different replication origin, namely the pBluescript's ori (ColE1 type). This "dual ori" strategy not only ensures stable coexistence of the two plasmids but may also offer performance advantages: maintaining the Plasmid A (sensor) at a lower copy number to reduce cellular metabolic burden, while allowing the Plasmid B (reporter) to remain at a higher copy number to amplify the final output signal.

3.System Integration and Final Verification:

The final step will be to removal the already verified sensing and reporter modules into these two newly designed backbones, pAZ2 (Plasmid A) and pBZ2 (Plasmid B), which have clear engineering objectives, and conduct comprehensive functional and safety verification of this final version of the biosensor.

---

**### Cycle 5: Custom Engineering of Plasmid Backbones and Preliminary Verification**

---

**Design**

After successfully verifying the core functionality of our biosensor in the fourth cycle, our engineering goal shifted from "functional realization" to "application optimization." We foresaw that in future co-culture detection of our engineered bacteria with the target bacterium (*P. aeruginosa*), to reduce contamination by other bacteria and increase the probability of false positives, we designed a bran-new, smarter two-plasmid system.

---

**Core Design Concept**

Introducing a Correlation Selection Marker: We decided to abandon general antibiotics and instead use *P. aeruginosa*'s own triclosan resistance gene (fabV) as the sole selection marker for both of our plasmids. This allows us to perform selection in media containing triclosan in the future, thereby inhibiting the growth of other bacteria and greatly reducing false positives.

Achieving Coexistence of Homologous Resistance Plasmids: How to make two plasmids both carrying fabV resistance coexist in the same host? We designed an ingenious "differential expression" strategy:

"Plasmid A" backbone (pAZ2): Sensing plasmid. Its fabV resistance gene is controlled by the arabinose-inducible promoter (pBAD).

"Plasmid B" backbone (pBZ2): Reporter plasmid. Its fabV resistance gene is controlled by a constitutive promoter (Pcat).

This design allows us to conveniently perform plasmid construction, transformation, and screening by controlling the presence or absence of arabinose.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e13.webp)

Figure 13. Recombinant Expression Plasmid pAZ2: Structure and Element Origins

![示例图片](https://static.igem.wiki/teams/5853/cycle/e14.webp)

Figure 14. Recombinant Expression Plasmid pBZ2: Structure and Element Origins

---

**Build**

1. Functional Verification of the fabV Gene: We first cloned the fabV gene from the genome of *P. aeruginosa* PAO1 and connected it to a constitutive promoter. Experiments proved that this gene successfully conferred triclosan resistance to E. coli, verifying the functionality of this component.

2. Construction and Verification of Inducible Resistance: Next, we placed fabV under the control of the pBAD promoter and, through induction/non-induction experiments, successfully proved that its resistance function is strictly regulated by arabinose.

3. De Novo Assembly of Plasmid Backbones: This was the core of this cycle. Through multi-fragment assembly, we successfully assembled the previously verified components—the ori replicons from pORI280 or pBlue, the fabV resistance module (inducible or constitutive), the strong T1T2 terminators from pBAD—to construct our brand new plasmid backbones:

pAZ2 (ori-araC-pBAD-fabV-T1T2)

pBZ2 (ori-Pcat-rbs-fabV-T1T2)

4. System Reassembly: Finally, we removal the four sensing modules and four reporter modules verified as perfectly functional in the fourth cycle onto our brand new pAZ2 and pBZ2 backbones, and then performed tests.

---

**Test**

The core goal of this test was "regression verification"—to verify whether the core functionality of our biosensor remained stable and efficient after switching to the brand new, complex plasmid backbones. We repeated the exact same induction experiment as in the fourth cycle.

Expected Result: The new system should perfectly reproduce the successful results of the fourth cycle.

Actual Result: The new system lived up to expectations and performed perfectly. The experimental results were highly consistent with those of the 4th round.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e15.webp)

Figure 15. Element test A: PqsR-PpqsA element test; B: SoxR-PmexG element test; C: BrlR-PbrlR element test; D: MexL-PphzA1 element test.

---

**Learn and Conclude**

The fifth cycle was a huge leap forward in the engineering depth of our project.

---

**Conclusion:**

We not only proved that our sensing-reporter modules are highly practical, but more importantly, we successfully tailored a set of new plasmid tools for the project. This new plasmid system is not only functionally reliable, but its built-in fabV selection system also clears obstacles for the future practical application of the project (such as detection during co-culture), greatly enhancing the application potential and innovativeness of our project.

---

**Next Step:**

We will further optimize the two-plasmid system, tandem multiple "sensor proteins," to increase its sensitivity for detecting *P. aeruginosa*.

---

**### Cycle 6: Multi-Module Sensing Combination and Culture System Optimization**

---

**Design**

Previous experiments indicated that the soxR-PmexG module produced the strongest signal in response to *P. aeruginosa*'s secondary metabolite PYO. Meanwhile, dry lab modeling results also predicted that this module had the best detection performance.

Therefore, we designed a composite sensing system, co-expressing pqsR and soxR, constructing a new Plasmid A (pAZ2: Pcat-RBS-pqsR-T0T1 + Pcat-RBS-soxR-T0T1), and simultaneously carrying both the PpqsA-*lacZ* and PmexG-gus reporter modules on the Plasmid B (pBZ2).

---

**Core Design idea**

To ensure the system can work in complex environmental water samples, we further designed experiments to optimize the culture medium and conditions to improve detection sensitivity and specificity.

---

**Build:**

The newly constructed Plasmid A and Plasmid B plasmids were introduced into the EC1000Δgus chassis strain, obtaining the two-plasmid engineered bacteria.

Based on literature and experimental results, explored the following optimization directions:

1.Culture Medium Screening: Compared the effect of PB medium vs. LB medium on PYO production by *P. aeruginosa*.

2. Composite Medium Construction: Combined PB with a commercial enzyme substrate medium (MMO-MUG) to form a system supporting the growth of both bacterial strains.

3. Small-Volume Culture Optimization: Attempted co-culture in 1.5 mL centrifuge tubes and 96-well plates to adapt to high-throughput detection needs.

4. Oxygen Supply Method Exploration: Included shaker, oxygen pump, exogenous chemical oxygenators (sodium bicarbonate, hydrogen peroxide, sodium percarbonate tablets, potassium nitrate) and breathable membrane materials (EPTFE) and architectural door/window nano-level breathable membranes.

---

**Test**

Composite Sensing System Detection: Capable of detection, but the fluorescence was not very strong in every well.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e16.webp)

Figure 16. Use engineering bacteria to test *P. aeruginosa* in domestic water and drinking water.

---

**Culture Medium Comparison:**

*P. aeruginosa* significantly produced PYO (green) in PB medium, while production was very low in LB. β-galactosidase activity assays further confirmed PB is superior to LB.

---

**Composite Medium Verification:**

PB+MMO-MUG can simultaneously support E. coli growth and PYO production by *P. aeruginosa*.

---

**Small-Volume Culture:**

In test tube systems, PYO was produced normally; but in centrifuge tubes and 96-well plates, bacterial growth was limited and PYO production was extremely low due to insufficient dissolved oxygen.

---

**Oxygenation Method Attempts:**

Both mechanical aeration and chemical oxygenators did not significantly increase PYO production and posed risks of contamination or toxicity; The breathable membrane material (EPTFE) significantly improved dissolved oxygen conditions while maintaining sterility, restoring PYO synthesis by *P. aeruginosa* in the 96-well plate system.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e17.webp)

---

**Learn and Conclude**

This cycle achieved two key breakthroughs:

1.Multi-Module Sensing Combination: Simultaneous use of pqsR and soxR improved the system's sensitivity and specificity for detecting *P. aeruginosa*.

2.Culture System Optimization: By selecting PB basal medium, introducing triclosan, using the PB+MMO-MUG composite system, and applying breathable membranes to improve small-volume oxygen supply, we established a stable high-throughput detection platform operable in 96-well plates.

These improvements provide complete experimental conditions for the final environmental water sample detection and also demonstrate the system's potential for accurate and rapid detection of *P. aeruginosa* in complex water bodies.

---

**Next Step:**

In subsequent cycles, based on the optimized system, we will conduct large-scale detection of different environmental water samples and compare the results with national standard methods to verify the system's accuracy and reliability.

---

**### Cycle 7: Final System Integration and Application Verification**

---

**Design**

Based on multiple rounds of optimization in the early stage, we have selected the dual-plasmid multi-module sensing system (with pqsR + soxR as sensors, and PpqsA–*lacZ* and PmexG–gus as dual reporters).

The optimized culture system (PB+MMO-MUG composite medium + triclosan selection pressure).

The 96-well plate system sealed with breathable membranes (solving the dissolved oxygen insufficiency problem).

To verify the practical application effect of the final system, we planned to detect different types of water samples and compare the results with the National Standard Method (GB method).

---

**Experiment:**

Detected water samples from different sources (lake water, tap water, purified drinking water, domestic water) in the 96-well plate system.

Simultaneously performed parallel detection of the same batch of water samples using the GB method.

---

**Detection:**

Actual Sample Detection:

In lake water and domestic water, the engineered bacteria detected significant color/fluorescence signals, indicating the presence of *P. aeruginosa*;

In purified drinking water and tap water, no color change occurred, indicating no target bacteria detected.

---

**GB Method Comparison:**

The detection results of the two methods were consistent across all samples, and the engineered bacteria detection showed advantages in color development time and operational convenience.

![示例图片](https://static.igem.wiki/teams/5853/cycle/e18.webp)

Figure 18. Actual Sample Detection

---

**Learn:**

The optimized culture system and breathable membrane strategy ensured the system's stability in small-volume, high-throughput environments.

The engineered bacteria detection was consistent with the GB method in accuracy, while having the advantages of faster response and simpler operation.

The system already has the potential for application in environmental water monitoring.

---

**Next Step:**

Future work will focus on the following aspects:

Further evaluate the system's stability and sensitivity in more complex real water samples (such as industrial wastewater, medical sewage).

Explore integrating the system with portable reading devices (e.g., smartphone cameras or small fluorescence detectors) to enhance on-site detection capability.

Improve biosafety design to ensure the system is safe and controllable in practical applications.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide
sufficient information for other teams to replicate our work.{% endblock %}
{% block header %}

![示例图片](https://static.igem.wiki/teams/5853/image/otherimage/52ffd90c9792637b24f5862a846d9616.webp)

{% endblock %}
{% block page\_content %}

## Methods

---

## 1. Materials

---

**1.1 Buffers & Media**

1×TAE Buffer (g/L):
Tris: 4.84 g.
 EDTA: 0.744 g.
 pH: Adjust the pH to 8.5 using glacial
acetic
acid.
 Bring the volume to 1 L with ddH₂O.

LB Medium (g/L):
NaCl: 10.0 g.
Yeast Extract: 5.0 g.
Tryptone: 10.0 g.
Agar: 15.0 g.

pH:
Adjust the pH to 7.0.
Autoclave at 121°C for 20 minutes.

LBS (Sucrose Selection) (g/L):
LB + Sucrose: 120.0 g.
pH: Adjust the pH to 7.0.
Autoclave at
121°C
for 20 minutes.

TSB Medium (g/L):
Tryptone Soya Broth: 30.0 g.
Agar: 15.0 g.
pH: Adjust the pH to 7.0.

Autoclave at 121°C for 20 minutes.

100 mM MgCl₂ Buffer (g/L):
MgCl₂: 9.521 g.
Dissolve in ultrapure water, then sterilize at 121°C
for 20
minutes using an autoclave.

100 mM CaCl₂ Buffer (g/L):
CaCl₂: 11.0984 g.
Dissolve in ultrapure water, then sterilize at 121°C
for
20 minutes using an autoclave.

100 mM Glycerol–CaCl₂ Buffer (g/L):
CaCl₂: 11.0984 g.
Glycerol: 100 mL.
Dissolve in ultrapure
water, then sterilize at 121°C for 20 minutes using an autoclave.

Sucrose Buffer (g/L):
Sucrose: 102.69 g.
Dissolve in ultrapure water, then sterilize at 121°C for
20
minutes using an autoclave.

---

**1.2 Antibiotics**

*E. coli*: Km 30 μg/mL; Cm 30 μg/mL; Tc 20 μg/mL; Gm 10 μg/mL; Amp 100 μg/mL.

P. aeruginosa: Km 30 μg/mL; Cm 30 μg/mL; Gm 100 μg/mL; Amp 100 μg/mL; Tc 200
μg/mL
(solid)/100 μg/mL (liquid).

---

**1.3 Culture Conditions**

37°C, 220 rpm unless stated otherwise.

---

## 2. Mutant Strain Construction

---

![示例图片](https://static.igem.wiki/teams/5853/safe/exfix1.webp)

**2.1 PCR amplification of DNA Fragments**

PCR was used to obtain the target DNA fragments. The PCR amplification system is shown in Table 1-1, and
the
PCR program is shown in Table 1-2.

**Table 1-1 PCR Reaction System**

| Component | Volume |
| --- | --- |
| DNA polymerase buffer | 25 μL |
| Primer F | 2 μL |
| Primer R | 2 μL |
| DMSO | 2.5 μL |
| 50% glycerol | 5 μL |
| DNA polymerase | 1 μL |
| Template | 2 μL |
| ddH2O | Up to 50 μL |

**Table 1-2 PCR Program**

| Temperature | Time |
| --- | --- |
| 95℃(pre-denaturation) | 5min |
| 95℃(denaturation) | 50s |
| Annealing temperature | 50s |
| 72℃(extension) | Extension time |
| 72℃(final extension) | 10min |
| 12℃(hold) | ∞ |

Note: The annealing temperature is determined by the F/R primers; the extension time is determined by the
length of the DNA fragment and the type of DNA polymerase used.

---

**2.2 Overlap PCR**

Two pairs of primers, up F/up R and low F/low R, were designed to amplify the upstream and downstream
sequences
of the target DNA fragment, respectively. Using *Escherichia coli EC1000*
genomic
DNA as the template, the upstream (UP) and downstream (LOW) sequences were amplified with the up F/up R
and low
F/low R primer pairs, respectively. These two amplicons were then used as templates for overlapping PCR
with the
up F/low R primer pair. The resulting PCR product was analyzed by agarose gel electrophoresis, and the
target
band was excised and purified to obtain the successfully fused UP–LOW fragment.

---

**2.3 Gel Extraction**

DNA Gel Purification and Recovery. The DNA gel extraction procedure was performed according to the
manufacturer's instructions for the DNA purification kit, with the following steps:

1. Activate the DNA adsorption column: Place the column into a collection tube, add 500 μL of BL buffer,
centrifuge at 12,000 rpm for 1 min, and discard the flow-through.

2. Weigh the DNA gel slice: Add PC buffer at a ratio of 100 μL per 0.1 g of gel. Incubate the tube in a
50°C
water bath until the gel is completely dissolved, gently inverting the tube periodically to accelerate
dissolution.

3. Load the sample: Transfer the entire solution to the adsorption column, centrifuge at 12,000 rpm for 1
min,
and discard the flow-through.

4. Wash the column: Add 600 μL of ethanol–PW buffer, centrifuge at 12,000 rpm for 1 min, discard the
flow-through, and repeat this step.

5. Dry the column: Centrifuge the empty column at 12,000 rpm for 2 mins, then place it into a new 1.5 mL
tube
and air-dry at room temperature for approximately 10 mins until all ethanol has evaporated.

6. Elute the DNA: Add an appropriate volume of pre-warmed (70°C) EB buffer to the column, centrifuge at
12,000
rpm for 2 mins, and collect the eluted target DNA fragment.

---

**2.4 Large-Scale Double Digestion**

Table 1-3 Double enzyme digestion reaction system

| Component | Volume |
| --- | --- |
| 10× digestion buffer | R µL |
| DNA fragment(plasmid) | 40 µL |
| Restriction endonuclease A | 4 µL |
| Restriction endonuclease B | 4 µL |
| ddH2O | Up to 200 µL |

R was determined by the type of restriction endonuclease. The condition of enzyme digestion is 37 ℃
reaction
for 8-16h.

**2.5 Digestion Clean-up**

Similar to gel extraction but add equal volume PC buffer to digestion mix before loading.

**2.6 Ligation**

Table 1-4Ligation system

| Component | Volume |
| --- | --- |
| 10×T4 DNA Ligase Buffer | 2 µL |
| Vector | 2 µL |
| T4 DNA Ligase | 1 µL |
| DNA fragment after enzyme digestion | 15 µL |

Incubate 16°C 8–16 h.

---

**2.7 Preparation of Competent E. coliont> Cells**

A single colony was picked from a freshly streaked E. coliont> growth plate
after
12 h of incubation, inoculated into fresh LB liquid medium without antibiotics, and cultured with
shaking for 12
hours.

The bacterial culture was then transferred at a 1% ratio into 50 mL of fresh LB liquid medium and grown
with
shaking until the logarithmic phase (OD₆₀₀ ≈ 0.4).

The bacterial suspension was transferred into a pre‑chilled 100 mL centrifuge tube, placed on ice for 30
mins,
centrifuged at 4,100 rpm for 10 mins at 4°C, and the supernatant was discarded.

Pre‑chilled 100 mM MgCl₂ buffer (10% of the original culture volume) was added to resuspend the cells,
followed
by incubation on ice for 10 mins. The suspension was centrifuged at 4,100 rpm for 10 mins at 4°C, and
the
supernatant was discarded.

Pre‑chilled 100 mM CaCl₂ buffer (4% of the original culture volume) was added to resuspend the cells,
followed
by incubation on ice for 30 mins. The suspension was centrifuged at 4,100 rpm for 10 mins at 4°C, and
the
supernatant was discarded.

Finally, pre‑chilled 100 mM glycerol–CaCl₂ buffer (4% of the original culture volume) was added to
resuspend
the cells. Aliquots of 100 μL were transferred into pre‑chilled 1.5 mL centrifuge tubes and stored at
−80°C.

Heat Shock Transformation of Competent E. coliont> Cells

3 μL of plasmid (20 μL from the DNA ligation reaction described in section 2.2.8) was added to the
competent
E. coliont> cells prepared above.

The transformation mixture was incubated on ice for 30 mins, immediately heat‑shocked in a 42°C water
bath for
90 s, and then returned to ice for 5 mins (temperature changes during this process should be rapid).

Approximately 0.8 mL of fresh LB medium was added to the transformation mixture, which was then incubated
with
shaking at 37°C for 60 mins (90 mins for ligation reaction mixtures).

Subsequently, about 100 μL of the bacterial suspension was evenly spread onto agar plates containing the
appropriate antibiotic (for ligation reaction mixtures, the cells were collected by centrifugation
before
plating). The plates were incubated upside‑down at 37°C.

---

**2.8 Recombinant Vector (or Plasmid) Extraction**

The recombinant vector (or plasmid) extraction procedure was performed according to the manufacturer's
instructions for the Plasmid Mini‑Prep Kit (Tiangen Biotech Co., Ltd.), with the following steps:

Transfer 5 mL of bacterial culture into a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 1 min
to
collect the cells.

Add 250 μL of RnaseA‑P1 buffer to the tube and resuspend the cells completely.

Add 250 μL of P2 buffer, gently invert to mix, and incubate to ensure complete cell lysis.

Add 350 μL of P3 buffer, gently invert to mix, and centrifuge at 12,000 rpm for 10 mins at 4°C.

Activate the adsorption column: Place the column into a collection tube, add 500 μL of BL buffer, and
centrifuge at 12,000 rpm for 1 min.

Transfer the entire supernatant into the adsorption column (avoiding the white precipitate), centrifuge
at
12,000 rpm for 1 min, and discard the flow‑through.

Add 500 μL of PD buffer, centrifuge at 12,000 rpm for 1 minute to remove proteins and discard the
flow‑through.

Add 600 μL of ethanol–PW buffer, centrifuge at 12,000 rpm for 1 min, discard the flow‑through, and repeat
this
step.

Centrifuge the empty column at 12,000 rpm for 2 mins, then place it into a new 1.5 mL tube and air‑dry at
room
temperature for approximately 10 mins until all ethanol has evaporated.

Add an appropriate volume of pre‑warmed (70°C) EB buffer to the column, centrifuge at 12,000 rpm for 2
mins,
and collect the eluted recombinant vector (or plasmid).

---

**2.9 Small‑Scale Double Digestion**

The reaction system for the small‑scale double digestion used in this study is shown in Table 1‑3.

---

**2.10 Conjugation**

The conjugative plasmid from section 2.8 was transformed into *Escherichia coli
S17‑1*
(hereinafter referred to as *S17‑1*) to serve as the donor strain,
with
*Pseudomonas aeruginosa* as the recipient strain.

Culture both the donor and recipient strains with shaking at 37°C and 220 rpm until reaching the
logarithmic
phase. Mix the donor and recipient cultures at volume ratios of 3:1 and 5:1 in sterile 1.5 mL centrifuge
tubes,
centrifuge at 5,500 rpm for 5 mins, and discard the supernatant.

Add 1 mL of fresh TSB liquid medium to the donor pellet, resuspend, transfer to the recipient pellet, mix
thoroughly, centrifuge at 5,500 rpm for 5 mins, and discard the supernatant.

Add another 1 mL of fresh TSB liquid medium to the mixture, resuspend, centrifuge at 5,500 rpm for 5
mins, and
discard the supernatant.

Add 100 µL of fresh TSB liquid medium to resuspend the cells, then spread the entire suspension onto a
non‑selective TSB agar plate. Incubate upright at 37°C for 2 days.

Resuspend the bacterial lawn with an appropriate amount of fresh TSB liquid medium, dilute, and spread
onto
selective agar plates containing the appropriate antibiotics. After approximately 2 days of incubation
at 37°C,
scrape the bacterial lawn, dilute appropriately, and spread onto the corresponding selective plates.

Heat Shock Transforma*E. coli*

3 μL of plasmid (20 μL from the DNA ligation reaction described in section 2.2.8) was added to the
competent E.
coli cells prepared above.

The transformation mixture was incubated on ice for 30 mins, immediately heat‑shocked in a 42°C water
bath for
90 s, and then returned to ice for 5 mins (temperature changes during this process should be rapid).

Approximately 0.8 mL of fresh LB medium was added to the transformation mixture, which was then incubated
with
shaking at 37°C for 60 mins (90 mins for ligation reaction mixtures).

Subsequently, about 100 μL of the bacterial suspension was evenly spread onto agar plates containing the
appropriate antibiotic (for ligation reaction mixtures, the cells were collected by centrifugation
before
plating). The plates were incubated upside‑down at 37°C.

---

## 3. *P. aeruginosa* Detection (National Standard Method)

---

**3.1 Water Sample Filtration**

All operations were performed in a Class 100 clean bench.

A 250 mL water sample was filtered through a 0.45 μm membrane filter.

The filter membrane was placed onto a CN agar plate, ensuring no air bubbles were trapped.

---

**3.2 Cultivation**

The plates were inverted and incubated at 36°C ± 1°C for 24–48 h.

---

**3.3 Result Observation and Colony Counting**

Colonies were observed at 20–24 h and again at 40–48 h.

Blue/green colonies (pyocyanin-producing) were counted and subjected to the pyocyanin test.

Fluorescent non-blue-green colonies were counted and subjected to the acetamide broth test.

Reddish-brown colonies were subjected to oxidase, acetamide, and King’s B tests.

---

**3.4 Confirmatory Tests**

Nutrient agar: Purification of suspected colonies.

Oxidase test: A purple color change within 10 s was considered positive.

King’s B medium: Fluorescence was examined under UV light.

Pyocyanin test: Extraction with chloroform followed by the addition of HCl; pink color was considered
positive.

Acetamide broth test: Nessler’s reagent was added; The color change from yellow to brick-red was
considered
positive.

---

**3.5 Counting and Reporting**

The count was calculated using the formula:

N=P+F(cFnF)+R(cRnR)N=P+F(nFcF)+R(nRcR)

The results were reported in CFU/250 mL.

---

---

## 4. Medium Optimization

---

**4.1 PB/MMO Mixed Medium**

Optimization included:

Ratio adjustment； Ion substitution；Carbon and nitrogen source combinations；Sodium bicarbonate/sodium
nitrate
gradients；Oxygenation treatments；Hydrogen peroxide addition；Oxygen-releasing tablet tests

---

**4.2 LB/TSB/BHI Comparison**

Addition of H₂O₂ and oxygen-releasing tablets; BHI was ultimately selected as the optimal medium.

---

**4.3 BHI Optimization**

UV-treated oxygen-releasing tablets；HEPES addition；Reporter gene plasmid introduction；Potassium nitrate
gradients

---

---

## 5. Experimental Validation

---

**5.1 Fluorescence Validation**

After constructing the target plasmid, it was transformed into pre-prepared *E.
coli*
via heat shock. The transformed bacterial suspension was spread onto LB plates containing the
appropriate antibiotic and incubated at 37°C overnight. Single colonies were picked and inoculated into
5 mL
liquid LB medium. The appropriate signaling molecule PQS and specific substrate ONPG, or the virulence
factor
PYO and specific substrate MUG, were added. The cultures were sealed in quantitative plates and
incubated at
37°C for 12–16 hours. After incubation, color changes were observed, and fluorescence was examined under
UV
light.

---

**5.2 Enzyme Activity Validation**

A single colony of the *Pseudomonas aeruginosa* strain to be tested was
inoculated into 5 mL liquid LB medium and cultured overnight at 37°C with shaking until reaching the
stationary
phase. OD₆₀₀ was measured. A 50 μL aliquot of the bacterial suspension was transferred to a 1.5 mL
centrifuge
tube, followed by the sequential addition of 420 μL Z Buffer, 20 μL chloroform, and 10 μL 0.1% SDS.
After
mixing, the tube was incubated in a 30°C incubator for at least 1 hour. Meanwhile, a 0.004 g/mL ONPG
substrate
buffer was prepared as required, fully dissolved, and also incubated at 30°C for 30 minutes. After
incubation,
100 μL of the prepared ONPG substrate buffer was added to each tube, timing was started immediately, and
the
tubes were returned to the 30°C incubator for 30–90 minutes. The reaction was terminated by adding 250
μL of 1
mol/L Na₂CO₃, and the reaction time was recorded. Finally, OD₄₂₀ and OD₅₅₀ of the reaction solution were
measured.

β-galactosidase activity was calculated using the formula:

Miller units =1000×（OD420–1.75×OD550）/[（culture OD600）×（Volume of culture）×（Reaction time）]

---

## References

---

1. Li Zhiying, Li Panxin, Ning Wanqing, et al. Analysis of the biological effects of the antABC
   operon in
   Pseudomonas aeruginosa. Acta Microbiologica Sinica, 2024, 64(11): 4403–4424.
2. Yan’an University, Yan’an Kelongni Biotechnology Co., Ltd. Pseudomonas aeruginosa fur gene
   deletion mutant
   and its construction method and application: 202311364298.
3. Wang Shuaitao, Gao Qianqian, Cheng Juanli, et al. Research progress on the composition of
   Pseudomonas
   aeruginosa biofilms and their regulation by quorum sensing systems and c-di-GMP. Acta
   Microbiologica Sinica,
   2021, 61(5): 1106–1122.
4. Wang Shuaitao, Niu Yanting, Zhang Heng, et al. Targeted protein delivery technology for
   disrupting
   Pseudomonas aeruginosa biofilms. Acta Microbiologica Sinica, 2021, 61(9): 2726–2748.
5. Wang Wenjuan, Chen Wenqing, Zhang Ming, et al. Detection and analysis of Pseudomonas aeruginosa
   in
   packaged drinking water. Quality Safety and Inspection and Testing, 2025, 35(3): 70–73.
6. Wang Yating. Preparation, characterization, and protective efficacy evaluation of a monoclonal
   antibody
   against Pseudomonas aeruginosa flagella. Master's thesis, Chongqing University of Technology,
   2025.
7. Shen Peiyao, Du Yaqiong, Xiao Youyu, et al. Comparative analysis and optimization of detection
   methods for
   Pseudomonas aeruginosa. Industrial Microbiology, 2024, 54(6): 121–123.
8. Chen Muyu, Zhu Sixia. Inhibitory effect of subinhibitory concentration of onion extract on
   quorum sensing
   in Pseudomonas aeruginosa. Neijiang Science & Technology, 2024, 45(10): 43–45, 77.
9. Xing Yun, Zhang Yan, Zhou Daohong, et al. Establishment of a rapid detection method for
   Pseudomonas
   aeruginosa based on real-time fluorescence recombinase polymerase amplification. International
   Journal of
   Laboratory Medicine, 2024, 45(19): 2329–2333.
10. Li Panxin, Cheng Juanli, Zhang Heng, et al. Research progress on the functional diversity of the
    quorum
    sensing signal molecule PQS in Pseudomonas aeruginosa. Acta Microbiologica Sinica, 2023, 63(9):
    3500–3519.
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    Res. 2025
    Oct 2;12(1):e003100.
12. Martínez-Rodríguez OP, García-Contreras R, Aguayo-Ortiz R, et al. Natural products inhibit
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    growth and modulate quorum-sensing systems Las and Rhl in Pseudomonas aeruginosa PA14. Chem
    Biodivers. 2025
    Oct 3:e02040.
13. Ashrafi B, Heydari R, Rezaei F, et al. Investigating the release of active compounds and
    cytotoxicity of
    thymol/gallic acid/β-cyclodextrin bio-nanocomposite: a targeted strategy with the approach of
    disrupting the
    genes involved in the quorum sensing system and biofilm formation in P. aeruginosa (PAO1). Prep
    Biochem
    Biotechnol. 2025 Oct 1:1–17.
14. Al-Helo FK, El-Banna N, Qaralleh H, et al. Quorum sensing inhibition and virulence factor
    attenuation in
    Pseudomonas aeruginosa by camphor. J Pharmacopuncture. 2025 Sep 30;28(3):229–239.
15. Falagas ME, Kontogiannis DS, Ragias D, et al. Antibiotics for the treatment of patients with
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16. Bellamoroso KR, Kostylev M, Smalley NE, et al. Inactivation of MexT in Pseudomonas aeruginosa
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    2025 Sep
    19:2025.09.19.677155.

{% endblock %}

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{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block header %}

![示例图片](https://static.igem.wiki/teams/5853/image/otherimage/project.webp)

{% endblock %}
{% block page\_content %}

### Detective Engineering Bacteria: Rendering *Pseudomonas aeruginosa* Undetectable in Water

---

**Abstract**

Based on synthetic biology strategies, this study successfully constructed an engineered *Escherichia coli* biosensor and established a high-throughput detection method using a 96-well quantitative plate for the specific detection of the opportunistic pathogen *Pseudomonas aeruginosa* in water. The sensor achieves high specificity through a triple-verification mechanism: recognition of the *Pseudomonas aeruginosa*-specific quorum-sensing molecule PQS, detection of the characteristic virulence factor pyocyanin (PYO), and selective enrichment via triclosan resistance. The reporting system employs the *lacZ* and *gus* genes, which catalyze the formation of yellow chromogenic and blue fluorogenic signals from the substrates ONPG and MUG, respectively. Results demonstrated that the engineered bacterial chassis exhibits a clean background with highly specific sensing elements capable of responding with high sensitivity to both PQS and PYO. In practical water sample detection, the method utilizes a 96-well quantitative plate format combined with the MPN (Most Probable Number) technique for accurate quantification. The results are in excellent agreement with those obtained using the national standard method, while the detection time is reduced from 48–72 hours to 6–12 hours. This study provides an accurate and practical new strategy for the rapid, low-cost on-site monitoring and high-throughput screening of *Pseudomonas aeruginosa*.

---

### 1. Validation of the *gus* Enzyme Substrate Detection Platform

The quantitative detection platform is based on a well-established enzyme substrate methodology, in which specific enzymatic activity of the target bacterium catalyzes colorless substrates to generate measurable chromogenic or fluorogenic products. To confirm the platform's applicability, we initially tested various water samples using a commercial enzyme substrate kit.

Samples included sterile water, wild-type *E. coli* suspension, environmental water samples, and commercially bottled water. Experiments were performed according to standard protocols using β-galactosidase (*lacZ*)- and β-glucuronidase (*gus*)-based commercial reagents [2]. Results demonstrated successful qualitative detection of *E. coli* in all sample types. The wild-type *E. coli* suspension and river water sample exhibited high *E. coli* counts, whereas sterile water and bottled water samples showed no detectable *E. coli* (Figure 1). This preliminary validation confirmed the stability and reliability of the enzyme substrate method across different water matrices, providing a robust methodological foundation for our detection system.

![示例图片](https://static.igem.wiki/teams/5853/safe/r1.webp)

**Figure 1. Validation of the enzyme substrate method applicability in various water samples.**

This figure presents the results of *Escherichia coli* detection in various water samples using a commercial enzyme substrate assay kit.

---

### 2. Genetic background engineering and biosafety containment validation of the EC1000 chassis strain

To ensure reporter signals originate exclusively from engineered plasmids rather than endogenous host activity, we constructed a clean chassis lacking both *lacZ* and *gus*. Additionally, to prevent plasmid dissemination into the environment, we incorporated a stringent physical containment system. *E. coli* EC1000 was selected as the parental strain due to its natural *lacZ* deficiency and genomic encoding of the RepA protein required for pORI280 replication, making it ideal for biosensor development. We then performed systematic genetic modifications and comprehensive functional validation.

We validated the endogenous β-galactosidase activity of EC1000 and its derivatives using both qualitative and quantitative approaches. Initially, blue–white screening was performed, which revealed that wild-type *Escherichia coli* ATCC 25922 (positive control) formed blue colonies, whereas EC1000 and EC1000 Δ*gus* colonies remained white (Figure 2A), indicating a lack of X-gal hydrolysis capability in the latter. For more precise confirmation, β-galactosidase activity was assessed using ONPG (o-nitrophenyl-β-D-galactopyranoside) as the substrate. Hydrolysis of ONPG by β-galactosidase yields yellow o-nitrophenol. As shown in Figure 2B, ATCC 25922 exhibited a distinct yellow coloration, whereas EC1000 and EC1000 Δ*gus* showed no significant color change.

However, the *gus* gene in the EC1000 genome remains active, which would interfere with fluorescence detection using *gus* as the reporter. To address this, we precisely deleted the *gus* gene from the EC1000 genome using a homologous recombination system, successfully generating the double-deletion strain EC1000 Δ*gus*. To confirm the deletion, EC1000 wild-type, EC1000 Δ*gus*, and ATCC 25922 were cultured in liquid medium containing MUG substrate and visualized under a 365 nm UV lamp. As shown in Figure 2C, EC1000 wild-type and ATCC 25922 emitted strong blue fluorescence, whereas the EC1000 Δ*gus* mutant produced no fluorescence, confirming successful *gus* deletion and providing a clean chassis with no background interference for the fluorescence reporting system.

To ensure that the genetic modifications did not adversely affect the basic physiological functions of the engineered bacteria, we determined the growth curves of the three strains. The results (Figure 2D) showed no significant differences in growth rate or final cell density between EC1000 Δ*gus* and its parental strain EC1000 or ATCC 25922, indicating that deletion of the *gus* gene did not impact normal bacterial growth and confirming the suitability of this chassis for subsequent sensor circuit integration and application.

To evaluate the effectiveness of the physical containment strategy based on the pORI280 conditional origin of replication, we electrotransformed test plasmids carrying the pORI280 origin of replication [3] (and containing triclosan resistance) into *E. coli* TG1, *E. coli* ATCC 25922, EC1000, and EC1000 Δ*gus*, using a vector with the pBlue origin of replication as a control. Spot plating results (Figure 2E) showed that the plasmid could only form colonies in EC1000 and its derivative EC1000 Δ*gus*, which provide the RepA replication protein, but failed to grow in TG1 and ATCC 25922. This result strongly demonstrates that our "lock-and-key" plasmid replication system strictly restricts plasmid replication to the engineered host strain, effectively preventing horizontal gene transfer to other environmental bacteria and thereby enhancing biosafety at the physical level.

In conclusion, we successfully obtained an engineered chassis strain EC1000 Δ*gus* with a clean genetic background (Δ*lacZ* Δ*gus*), normal growth characteristics, and stringent biosafety containment, providing a solid foundation for the construction of high-performance biosensors.

![示例图片](https://static.igem.wiki/teams/5853/safe/r2.webp)

**Figure 2. Validation of genetic engineering and biosafety containment of the chassis strain.**

(A) Blue–white screening results showed that wild-type *Escherichia coli* ATCC 25922 (positive control) formed blue colonies, whereas the engineered chassis strains EC1000 and EC1000 Δ*gus* formed white colonies, indicating the absence of endogenous β-galactosidase activity. (B) The ONPG liquid colorimetric assay further confirmed that only ATCC 25922 could hydrolyze ONPG to produce a yellow product, whereas the EC1000 series strains exhibited no such activity. (C) The MUG fluorescence assay, observed under ultraviolet light, showed that EC1000 wild-type and ATCC 25922 emitted blue fluorescence, while the EC1000 Δ*gus* mutant produced no fluorescence, confirming successful deletion of the *gus* gene. (D) Growth curve analysis indicated no significant difference in growth rate between the engineered strain EC1000 Δ*gus* and its parental strain, demonstrating that the genetic modification did not affect normal physiological functions. (E) Physical containment validation experiments showed that the test plasmid based on the pORI280 conditional origin of replication could only grow in engineered host strains (EC1000 and its derivatives) that provide the RepA protein, confirming that this system effectively prevents horizontal plasmid transfer.

---

### 3. Selective Pressure System Validation

To ensure high specificity of the detection system in complex microbial environments, we developed a selective pressure system based on dual antibiotics (triclosan and kanamycin). The primary objectives were: first, to verify that the triclosan resistance gene *fabV* had been successfully introduced into the chassis strain and was functioning effectively; and second, to demonstrate that triclosan could efficiently eliminate the vast majority of common environmental bacteria, allowing only the engineered strain and the target bacterium—*Pseudomonas aeruginosa*—to survive.

To achieve these goals, nine representative strains were selected, including the engineered strain, multiple control strains, and common environmental bacteria (EC1000Δ*gus* Pcat-*fabV* [engineered strain], S17-1, TG1, *E. coli* ATCC 25922, EC1000Δ*gus*, EC1000, *Salmonella enterica*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* PAO1). Spot plating experiments were performed on LB agar plates with and without triclosan (6 μg/mL). The results, as shown in Figure 3, demonstrated that on triclosan-containing plates, only the engineered strain carrying the *fabV* gene (EC1000Δ*gus* Pcat-*fabV*) and the naturally tolerant *Pseudomonas aeruginosa* PAO1 were able to grow. In striking contrast, the parental strains EC1000Δ*gus* and EC1000, as well as all other tested strains, were completely inhibited. This result conclusively confirmed that the *fabV* resistance gene had been successfully integrated into the engineered strain and was functioning as intended, thereby achieving the genetic modification objectives for the chassis. Furthermore, all other tested strains, including common pathogens such as *Salmonella enterica* and *Staphylococcus aureus*, as well as other *Escherichia coli* strains that may be present in the environment (e.g., S17-1, TG1, ATCC 25922)—were effectively eliminated.

In conclusion, this experiment not only successfully validated the functional expression of triclosan resistance in the engineered strain but also confirmed that the antibiotic-based strategy could create a highly specific selection environment, minimizing false-positive interference and providing a fundamental guarantee for the specificity and reliability of subsequent biosensing detection.

![示例图片](https://static.igem.wiki/teams/5853/safe/r3.webp)

**Figure 3. Validation of the triclosan-based selective pressure system.**

Spot plating experiments compared the growth of nine representative strains on LB agar plates without antibiotics (left) and with triclosan (6 μg/mL, right). The results clearly showed that on triclosan-containing plates, only the engineered strain carrying the *fabV* resistance gene (EC1000Δ*gus* Pcat-*fabV*) and the naturally tolerant *Pseudomonas aeruginosa* PAO1 were able to grow, while all other tested strains were completely inhibited. This demonstrates the effectiveness and high specificity of the selective pressure system.

---

### 4. Vector Element Validation

To ensure that the function of each genetic element in the biosensor system meets the design expectations, we conducted functional validation of both the promoter elements in the sensing circuit and the inducible resistance system.

4.1 Sensor Element Specificity

In addition to the resistance elements validated in section 1.3, the core of the sensing circuit lies in the promoters that specifically respond to PQS or PYO. To ensure the correct functionality of the promoters (PpqsA, PmexG, PphzA1, and PbrlR) selected from *Pseudomonas aeruginosa*, we first verified their activity within the native *P. aeruginosa* host.

The aforementioned promoters were individually ligated to a promoterless *lacZ* reporter gene to construct recombinant plasmids, which were then electrotransformed into *P. aeruginosa* PAO1. Subsequently, exogenous PQS or PYO was added to the culture medium for induction, and β-galactosidase activity (Miller Units) was measured to quantify promoter strength. The results showed that the PpqsA promoter activity was significantly upregulated in the presence of PQS, while PmexG, PphzA1, and PbrlR exhibited varying degrees of activation under PYO induction (Figures 4A–D). This experiment confirmed the correct functionality and induction specificity of these promoter elements in their native host, thereby providing reliable components for subsequent sensing circuit construction in engineered bacteria.

![示例图片](https://static.igem.wiki/teams/5853/safe/r4.webp)

**Figure 4. Functional specificity validation of sensor elements (promoters) in the native host.**

Candidate promoters (PpqsA, PmexG, PphzA1, and PbrlR) derived from *Pseudomonas aeruginosa* were ligated to the *lacZ* reporter gene and introduced into *P. aeruginosa* PAO1. The response specificity was quantified by measuring β-galactosidase activity (Miller Units). (A) PpqsA promoter activity was significantly upregulated following the addition of its signaling molecule PQS. (B–D) PmexG, PphzA1, and PbrlR promoter activities were all effectively activated upon the addition of their signaling molecule PYO. This experiment confirmed the correct functionality of these promoters in their native host.

4.2 Functional Validation of the Inducible Resistance System

To achieve precise control of the engineered bacteria and enhance their reliability in practical applications, we introduced a triclosan resistance gene (*fabV*) regulated by an arabinose-inducible promoter (pBAD) into the plasmid system. The design is such that *fabV* is expressed only in the presence of arabinose, conferring triclosan resistance; in the absence of arabinose, no resistance is expressed, and the bacteria cannot grow in a triclosan-containing environment.

To validate the system, the plasmid carrying the pBAD-*fabV* expression cassette was introduced into the EC1000 Δ*gus* strain, and spot plating experiments were performed on LB agar plates containing triclosan (3 μg/mL) with or without arabinose (0.1%). The results, as shown in Figure 5, demonstrated that the engineered bacteria grew normally in the presence of arabinose, whereas no colonies formed in the absence of arabinose. This clearly indicates that the arabinose promoter effectively regulates *fabV* expression, thereby achieving inducible control of exogenous resistance.

The successful construction of this inducible resistance system not only provides a more flexible means of selectively enriching engineered bacteria in complex environments but also further reduces the environmental safety risks associated with gene leakage.

![示例图片](https://static.igem.wiki/teams/5853/safe/r5.webp)

**Figure 5. Functional validation of the arabinose-inducible triclosan resistance system.**

A: Arabinose-inducible triclosan resistance system.

B: The engineered bacteria carrying the pBAD-*fabV* system were spotted on LB agar plates containing triclosan (3 μg/mL) with or without 0.1% arabinose. The results showed that the engineered bacteria grew normally only on the plates supplemented with arabinose, whereas no growth was observed in the absence of arabinose, confirming the stringent regulatory function of the inducible resistance system.

---

### 5. Detection System Vector Construction

After completing the functional validation of each vector element (including promoters, reporter genes, and resistance systems), we proceeded to construct the vector backbone for the final detection system. This backbone was designed to integrate both the PQS-sensing module and the PYO-sensing module, ensuring their stable coexistence without mutual interference, while retaining essential selective pressure and biosafety control elements.

A modular assembly strategy was adopted to construct two core plasmids: pAZ2 and pBZ2. The pAZ2 plasmid utilizes the conditional origin of replication from pORI280, whose replication strictly depends on the RepA protein provided by the engineered host strain EC1000 Δ*gus*, thereby effectively preventing the leakage of the plasmid into environmental strains.

The vector construction process is schematically illustrated in Figures 6A and 6B. Each functional module was sequentially assembled using restriction enzyme digestion and ligation, and the correctness and orientation of all elements were verified by DNA sequencing.

The successful construction of this dual-plasmid backbone provides a stable and controllable genetic platform for subsequent dual-signal detection of PQS and PYO, and lays the foundation for further optimization of detection performance.

![示例图片](https://static.igem.wiki/teams/5853/safe/r6.webp)

**Figure 6. Schematic illustration of the construction of the dual-plasmid sensing system vectors.**

A: Sensing plasmid pAZ2； B: Reporter plasmid pBZ2.

---

### 6. PQS Sensing System Validation

To achieve efficient detection of the *Pseudomonas aeruginosa*-specific quorum-sensing molecule PQS, we constructed a sensing circuit based on the PqsR protein and its cognate promoter PpqsA. This circuit uses *lacZ* as the reporter gene, whose expression product—β-galactosidase—catalyzes the hydrolysis of the colorless substrate ONPG to produce yellow o-nitrophenol, thereby enabling visual detection of the PQS signal (Figure 7A).

To validate the functionality of this circuit, the engineered bacteria harboring the PqsR–PpqsA sensing module were exposed to various concentrations of PQS, and the ONPG substrate was added to initiate the color reaction. The results, as shown in Figure 7B, demonstrated that in the PQS-treated groups, the solution developed a distinct yellow color over the course of incubation, whereas the control group without PQS remained colorless. This result clearly indicates that the PqsR–PpqsA sensing circuit can be specifically activated by PQS, successfully driving the expression of the *lacZ* reporter gene and ultimately generating an easily interpretable detection signal through a visible color change.

In conclusion, the PqsR–PpqsA sensing circuit efficiently and specifically responds to the PQS signal and achieves detection through an intuitive color change, providing a reliable PQS-sensing module for the subsequent construction of a complete dual-signal detection system.

![示例图片](https://static.igem.wiki/teams/5853/safe/r7.webp)

**Figure 7. Colorimetric validation of the PQS sensing system.**

A: Schematic illustration of the PqsR–PpqsA sensing circuit. B: The engineered bacteria carrying the PqsR–PpqsA sensing circuit were incubated in culture medium containing the ONPG substrate and exposed to PQS (right) or DMSO as a control (left). The results showed that only the PQS-treated group developed a yellow color, indicating successful activation of the β-galactosidase reporter pathway and confirming the chromogenic detection of the PQS signal.

---

### 7. PYO Sensing System Optimization and Validation

To optimize the detection performance for pyocyanin (PYO), a characteristic virulence factor of *Pseudomonas aeruginosa*, we implemented the “choose-one-from-multiple” strategy proposed during the design phase and conducted a systematic comparative analysis of three parallel PYO-sensing circuits. These circuits were constructed using different transcriptional regulatory proteins (SoxR, MexL, BrlR) and their corresponding specific promoters (PmexG, PphzA1, PbrlR). Through molecular docking simulations and experimental validation, our objective was to identify the sensing module with the best response performance.

**7.1 Molecular Docking Analysis**

We first employed molecular docking to simulate the binding modes and affinities between PYO and the key regulatory proteins (SoxR, MexL, BrlR) in the three circuits. The docking results, as shown in Figure 8, indicated that the regulatory protein BrlR exhibited the lowest binding energy with PYO (−7.26 kcal/mol), suggesting the strongest theoretical binding affinity. The binding energies of MexL and SoxR with PYO were −7.10 kcal/mol and −5.79 kcal/mol, respectively, showing a gradual decrease in theoretical affinity.

Although molecular docking suggests that BrlR may have an advantage at the protein–ligand binding level, transcriptional activation efficiency is influenced by multiple factors, including protein expression levels, the intracellular environment, and the intrinsic strength of the promoter.

![示例图片](https://static.igem.wiki/teams/5853/safe/r8.webp)

**Figure 8. Molecular docking analysis of the binding modes and affinities between PYO and various regulatory proteins.**

7.2 Experimental Detection Performance Comparison

To evaluate the performance of the three circuits in the actual sensing system, we introduced the complete sensing modules—SoxR–PmexG, MexL–PphzA1, and BrlR–PbrlR—into the engineered bacteria. Under induction with the same concentration of PYO, their performance was assessed by quantitatively measuring the fluorescence intensity generated by the *gus* reporter gene. The working principles are illustrated in Figures 9A–C.

The results, as shown in Figure 9D, indicate that the SoxR–PmexG circuit exhibited the highest fluorescence signal intensity and the lowest background noise in practical detection, demonstrating the best sensitivity and reliability. The MexL–PphzA1 circuit displayed basal fluorescence in the absence of PYO, and this fluorescence was significantly reduced upon the addition of PYO (dissolved in DMSO), representing a typical repressive response pattern; however, the extent of reduction was not particularly pronounced. Although the BrlR–PbrlR circuit showed the most favorable binding energy in molecular docking, it produced the weakest reporter signal in the actual system, possibly due to insufficient protein expression or lower transcriptional activation efficiency.

![示例图片](https://static.igem.wiki/teams/5853/safe/r9.webp)

**Figure 9. Performance comparison of different PYO sensing circuits in actual detection.**

A: Working principle of the SoxR–PmexG sensing circuit. B: Working principle of the MexL–PphzA1 sensing circuit. C: Working principle of the BrlR–PbrlR sensing circuit. D: Fluorescence intensity generated by the *gus* reporter gene in different sensing circuits under induction with the same concentration of PYO.

Integrating the results of molecular docking and experimental validation, we conclude that although the BrlR protein exhibits the highest theoretical binding capacity, the actual performance of its associated circuit is suboptimal. The MexL–PphzA1 circuit demonstrates a repression-type response profile, making it suitable for negative verification under specific contexts. In contrast, the circuit composed of SoxR–PmexG exhibits the most excellent sensitivity and reliability in practical detection. Therefore, the SoxR–PmexG module is selected as the core component for PYO sensing in subsequent sensing system development.

---

### 8. Screening of Sealing Films for 97-Well Quantitative Plates Based on Oxygen Supply Optimization

The production of the quorum-sensing molecule PQS and the virulence factor pyocyanin (PYO) by *Pseudomonas aeruginosa* occurs during the late growth phase at high cell density and is strictly aerobic. However, under conventional sealed culture conditions, dissolved oxygen in the medium is rapidly depleted before the late growth phase, severely limiting the synthesis of PQS and PYO and thereby compromising detection reliability. To address the insufficient dissolved oxygen supply during stationary culture—particularly in the late growth phase—which restricts the production of the key signaling molecules PQS and PYO, we screened and optimized the sealing film materials for 97-well quantitative plates.

In this study, five materials were compared for their performance: EPTFE, nanoscale breathable film for architectural doors and windows, PVDF, PTFE, and TPU. The results showed that EPTFE film exhibited the best performance in maintaining a sterile environment while supporting efficient oxygen transfer. Cultures sealed with EPTFE developed the characteristic green coloration indicative of PYO production. PVDF film supported partial PYO synthesis but resulted in unstable culture conditions due to water evaporation. In contrast, PTFE and TPU films failed to support PYO production effectively due to insufficient gas permeability (Figure 10).

In conclusion, by adopting the highly breathable EPTFE sealing film, we successfully established a reliable system suitable for small-volume, high-throughput cultivation, providing critical technical support for subsequent PYO-based sensing and detection.

![示例图片](https://static.igem.wiki/teams/5853/safe/r10.webp)

**Figure 10. Screening of sealing membrane materials for 96-well quantitative plates based on oxygen supply optimization.**

CK: Control group using the 97-well plate's original heat-sealable aluminum foil film.

a–e: Experimental groups using polyvinylidene fluoride (PVDF) membrane, polytetrafluoroethylene (PTFE) membrane, expanded polytetrafluoroethylene (EPTFE) membrane, nanoscale breathable film for architectural doors and windows, and thermoplastic polyurethane (TPU) film, respectively.

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### 9. Construction of the Engineered Bacteria and Validation of Dual-Response to Target Signaling Molecules

Following the successful validation of each individual component, we constructed the complete dual-plasmid sensing vectors, designated pAZ2-1 and pBZ2-1, which contain the PQS-sensing circuit and the PYO-sensing circuit, respectively (Figure 11).

![示例图片](https://static.igem.wiki/teams/5853/safe/r11.webp)

**Figure 11. Schematic diagram of the dual-plasmid sensing vector**

A: Schematic representation of the pAZ2 vector map.

B: Schematic representation of the pBZ2 vector map.

Since both plasmids carry the triclosan resistance gene *fabV*, sequential transformation into the EC1000 Δ*gus* host presented a conflict in antibiotic selection. To resolve this issue, we leveraged the arabinose-inducible expression of *fabV* in pAZ2. First, under arabinose-supplemented conditions, we successfully introduced pAZ2-1 into EC1000 Δ*gus* via triclosan resistance selection. Subsequently, in the absence of arabinose, we utilized the constitutively expressed *fabV* in pBZ2-1 to perform a second triclosan resistance selection, introducing pBZ2-1 into the engineered strain already harboring pAZ2-1. This process yielded the final engineered strain carrying the complete dual-plasmid system. Following the introduction of the complete dual-plasmid system (comprising the PQS-sensing circuit and the PYO-sensing circuit) into the EC1000 Δ*gus* host, the final detection strain, designated EC1001, was successfully constructed. Its working principle is illustrated in Figure 12.

![示例图片](https://static.igem.wiki/teams/5853/safe/r12.webp)

**Figure 12. Schematic diagram of the working principle of the EC1001 detection system**

To verify whether EC1001 functions according to the designed logic—i.e., responding to PQS and PYO separately and producing corresponding signals—mid-logarithmic phase cultures of the engineered strain were aliquoted into three groups of test tubes: one supplemented with DMSO, one with PQS (dissolved in DMSO), and one with PYO. All tubes were simultaneously added with the substrates ONPG and MUG, followed by incubation at 37°C in the dark. Observations were made after 10 hours of incubation.

The results, as shown in Figure 13, demonstrated that the PQS-treated group developed a yellow color (indicating the hydrolysis of ONPG by β-galactosidase) but exhibited no significant fluorescence under UV light. In contrast, the PYO-treated group showed no color change but emitted strong blue fluorescence under UV light (indicating the hydrolysis of MUG by β-glucuronidase). The DMSO control group showed neither color nor fluorescence changes throughout the experiment.

These results confirm that the engineered strain can specifically distinguish between and respond to PQS and PYO signals, producing observable colorimetric and fluorescent outputs through independent reporter pathways. This successfully achieves the "dual verification" logic as designed.

![示例图片](https://static.igem.wiki/teams/5853/safe/r13.webp)

**Figure 13. Dual-response validation of the engineered bacteria to target signal molecules.**

The engineered bacteria were exposed to DMSO (solvent control), PQS, and PYO, respectively, with the simultaneous addition of ONPG (chromogenic substrate) and MUG (fluorescent substrate) for incubation. Upper panel (visible light): Only the PQS-treated group developed a yellow color, indicating that the β-galactosidase reporter pathway was specifically activated. Lower panel (UV light): Only the PYO-treated group emitted strong blue fluorescence, indicating that the β-glucuronidase reporter pathway was specifically activated. The DMSO control group showed no signal throughout the experiment. These results directly confirm that the engineered strain can distinguish between PQS and PYO and performing "dual verification" as designed.

### 10. Performance Evaluation in Actual Water Samples: Comparison with the National Standard Method

The ultimate goal of this study was to develop a highly accurate biosensor system. To evaluate the engineered bacterial detection method, we selected five representative water samples: laboratory tap water, office drinking water, swimming pool water, residential building tap water, and sterile water. Each sample was divided into two aliquots. One aliquot was tested using the 97-well quantitative plate method developed in this project (containing engineered bacteria, substrates, and triclosan), incubated at 37°C for 12 hours, and the number of dual-signal positive wells was recorded (Figure 14A). The concentration was then calculated using the Most Probable Number (MPN) method. Figure 14B presents the MPN lookup table for converting the number of positive wells into bacterial concentration. The other aliquot was tested in strict accordance with the national standard method (GB/T 8538-2016, membrane filtration method).

The calculation of results using the national standard method included the enumeration and confirmation of both typical and atypical colonies. The formula for calculating the total number (N) of confirmed *Pseudomonas aeruginosa* colonies on the filter membrane is:

N = P + F(cF / nF) + R(cR / nR)

where:

P = number of confirmed positive typical colonies

F and R = total number of typical and atypical colonies on the filter membrane, respectively

cF and cR = number of colonies selected for confirmation from the typical and atypical colonies, respectively

nF and nR = number of confirmed positive colonies among the selected typical and atypical colonies, respectively

The final concentration is reported as CFU/250 mL, calculated using the formula:

(N × 250) / Volume of filtered water (mL)

Table 1 presents the detailed raw data (P, F, cF, nF, R, cR, nR) and final calculated results for each water sample.

The quantitative comparison between the two methods is shown in Figure 14C. Statistical analysis indicated no significant difference between the detection results of the engineered bacterial method and the national standard method (p > 0.05). This demonstrates that the biosensor system maintains the same level of accuracy as the "gold standard" method while reducing the detection time from 48–72 hours to less than 12 hours, thereby exhibiting excellent potential for on-site applications.

Table 1: Raw data and quantitative results for *Pseudomonas aeruginosa* detection in different water samples using the national standard method (GB/T 8538-2016)

| Water body | The colony number of Bacteria | P | F | cF | nF | R | cR | nR | The colony number of PA CFU/250ml |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Laboratory tap water | 81 | 2 | 12 | 3 | 12 | 27 | 6 | 18 | 7.25 |
| Office drinking water | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Swimming pool water | 63 | 1 | 12 | 1 | 6 | 43 | 1 | 20 | 5.15 |
| Residential building tap water | 112 | 1 | 18 | 2 | 6 | 83 | 2 | 20 | 15.3 |
| Sterile Water | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |

![示例图片](https://static.igem.wiki/teams/5853/safe/r14.webp)

**Figure 14. Comparison of actual water sample detection performance with the National Standard Method.**

(A) Schematic representation of the 96-well plate results for detecting different water samples using the engineered bacterial method developed in this project (photographed under visible and UV light). The color or fluorescence change in each well represents the result of one detection unit. (B) MPN (Most Probable Number) lookup table used to convert the number of positive wells into bacterial concentration. (C) Comparative graph of quantitative detection results for *Pseudomonas aeruginosa* in five water samples using the engineered bacterial method and the national standard method (GB/T 8538-2016). Statistical analysis indicates no significant difference between the results of the two methods (p > 0.05), while the engineered bacterial method reduces the detection time from 48–72 hours (national standard method) to less than 12 hours.

Conclusion and Discussion

In this study, we successfully constructed and systematically validated a highly specific, rapid, and quantitative whole-cell biosensor for the detection of *Pseudomonas aeruginosa* in water. Based on synthetic biology strategies, this sensor achieves highly specific identification of the target bacterium through a triple-verification mechanism—sensing the quorum-sensing molecule PQS unique to *P. aeruginosa*, detecting the characteristic virulence factor pyocyanin (PYO), and enabling selective enrichment through triclosan resistance. The reporting system employs the *lacZ* and *gus* genes to produce chromogenic and fluorescent signals, respectively, offering the advantages of intuitive, dual-channel verification.

Through rigorous genetic engineering, we obtained the engineered chassis strain EC1000 Δ*gus*, which exhibits a clean background (Δ*lacZ* Δ*gus*), normal growth, and strict biosafety containment capabilities. This effectively avoids endogenous enzyme interference and the risk of environmental dissemination of engineered plasmids. Validation of the selective pressure system further ensures high specificity and anti-interference capability of the detection system in complex microbial environments.

In actual water sample testing, the results obtained by this method were highly consistent with those of the national standard method (GB/T 8538-2016), showing no significant difference, while the detection time was dramatically reduced from 48–72 hours to 6–12 hours, significantly improving detection efficiency. Furthermore, this method requires no complex instrumentation, is cost-effective, and simple to operate, demonstrating excellent potential for on-site applications.

In conclusion, this study not only provides an accurate, rapid, and low-cost new strategy for detecting *P. aeruginosa*, but also offers a viable model for the application of synthetic biology in environmental microbial monitoring. In the future, further optimization of signal amplification circuits and the development of portable reading devices could promote the industrial application of this biosensor in practical water management, public health monitoring, and other scenarios.

References

[1] Flores SS, Clop PD, Barra JL, Argaraña CE, Perillo MA, Nolan V, Sánchez JM. His-tag β-galactosidase supramolecular performance. Biophysical chemistry, 2022, 281: 106739.

[2] Sarhan HR, Foster HA. A rapid fluorogenic method for the detection of *Escherichia coli* by the production of beta-glucuronidase. The Journal of applied bacteriology, 1991, 70(5): 394-400.

[3] Leenhouts K, Buist G, Bolhuis A, ten Berge A, Kiel J, Mierau I, Dabrowska M, Venema G, Kok J. A general system for generating unlabelled gene replacements in bacterial chromosomes. Molecular & general genetics : MGG, 1996, 253(1-2): 217-224.

Despite the significant achievements of this study, certain limitations remain. For instance, although the engineered bacterial method demonstrates high consistency with the national standard method in actual water sample detection, its sensitivity for detecting extremely low concentrations of *Pseudomonas aeruginosa* still requires further improvement. Future research could consider optimizing the design of sensing elements to enhance their responsiveness to low concentrations of signaling molecules.

Furthermore, this study only verified the biosensor's performance in several common types of water samples. Its applicability to more complex aquatic environments, such as industrial wastewater containing high levels of organic matter or heavy metals, still needs to be further evaluated. Addressing these issues will require exploring the introduction of more efficient signal amplification mechanisms or implementing targeted modifications to the engineered bacteria to enhance their stability and detection performance in complex environments.

Additionally, more extensive testing with various types of water samples should be conducted to comprehensively assess the biosensor's application range. Through continuous improvement and optimization, it is expected that the application scenarios of this biosensor can be further expanded, providing more powerful technical support for environmental microbial monitoring.

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