Experimental Progress
March
March 12, 2025 – Project Initiation and Chassis SelectionObjective: 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
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
March 20, 2025 – Δgus Gene Knockout
Objective: To construct a zero-background chassis strain lacking the gus gene (E. coli EC1000Δgus) and eliminate background interference from the endogenous gus gene in subsequent experiments.
Methods and Procedures:
A gus deletion (Δgus) 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 Δgus fragment and the EC1000 genome.
Results: The E. coli EC1000Δgus 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
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
April
April 3, 2025 – Reporter Module ConstructionObjective: 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
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
Cycle 1 Summary (March 12 – April 7)
Phase Achievements:
Chassis strain modification completed: Successfully constructed the zero-background chassis strain E. coli EC1000Δgus, 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
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 EC1000Δgus.
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
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
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
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
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
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
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 EC1000Δgus.
The previously constructed control plasmids were simultaneously transformed into EC1000Δgus 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
May
May 2, 2025 – Functional Validation ExperimentObjective: 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
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
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
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
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
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
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
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
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
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
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 EC1000Δgus.
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
June
June 1, 2025 – Functional Validation ExperimentObjective: 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
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
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
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
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
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
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
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
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
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
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
July
July 1, 2025 – System AssemblyObjective: 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 EC1000Δgus.
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
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
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
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
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 – August 15, 2025
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
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
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
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 ReconstructionObjective: 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 – August 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: August 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 EC1000Δgus.
Methods and Procedures:
Used electroporation to simultaneously introduce composite PAZ2-1 and composite PBZ2-1 into EC1000Δgus.
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 SetupObjective: 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 (August 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 SummaryObjective: 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.