示例图片

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

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

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 EC1000ΔgusA 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 EC1000ΔgusA.

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 EC1000ΔgusA chassis, obtaining all test strains and their corresponding negative control strains.

示例图片

Figure 3. Fluorescence phenotypic verification of EC1000 ΔgusA gene knockout.

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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

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

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 EC1000ΔgusA.

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

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

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 EC1000ΔgusA chassis again, preparing for the final verification.

3.

示例图片

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

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

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

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

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

示例图片

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

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

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

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

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.