Module1- Design

Engineering Bacteria Selection: "Model Verification" to "Application Implementation"

Bifidobacterium longum is the ultimate target strain for engineered strain modification in this project, owing to its excellent biological safety, physiological compatibility with the host, and intestinal colonization capacity. However, the experimental manipulation system of Bifidobacterium still has notable limitations, including insufficient genetic background characterization and low transformation efficiency. If directly used to establish the early-stage verification system, it may result in unstable experimental processes and a significant extension of the development cycle.

To reduce the early-stage technical barrier and ensure the reliability of core system function verification, we prioritize using E.coli as the model strain, leveraging its mature experimental manipulation system, including high-efficiency transformation, well-characterized genetic background, and a comprehensive tool enzyme repertoire. we complete the construction and functional debugging of the verification system, thereby laying a foundation for subsequent migration to Bifidobacterium longum.

Plasmid Compatibility Selection

Through literature research we ultimately selected pMB1-A as the vector plasmid to achieve a seamless transition from "E. coli verification to Bifidobacterium application". Its key advantage is its design as a shuttle plasmid specifically tailored for Bifidobacterium and E. coli, which facilitates both experimental manipulation and functional validation in both strains. This eliminates the need to reconstruct vectors for different bacterial species, significantly improving the system's transfer efficiency while ensuring the continuity of experimental processes and the reliability of results.

During the vector construction stage, we employed a homologous recombination enzyme-based approach to precisely insert target gene fragments into the pMBI-A plasmid. We also selected XbaI and EcoRI as double restriction enzyme sites, thereby establishing a coordinated "restriction digestion--recombination" vector construction system.

Function realization

We introduce the β-galactosidase (bgaB) from Bacillus thermoproteolyticus into the engineered strain to construct a functional system centered on "lactose substrate input-catalytic signal response". The core regulatory elements consist of a dual-component module:"lactose transporter LacY + optimized catalytic enzyme bgaB". Notably, bgaB was subjected to optimization via dry experiments, which significantly enhanced its adaptability to the intestinal environment and lactose catalytic efficiency. This optimization lays a critical molecular foundation for the system to achieve efficient lactose utilization and trigger downstream therapeutic functions.

Primary consideration was given to the fact that Bifidobacterium relies on a specific transport system for lactose absorption, and exhibits limited lactose uptake in its native state owing to insufficient competitive capacity. To address this, the design enhances lactose acquisition by introducing lactose permease (lacY): As a key substrate transport component, LacY (encoded by lacY) can specifically mediate the transport of intestinal lactose across the bacterial cell membrane into the cytoplasm. This directly mitigates the inherently low lactose transport efficiency of Bifidobacterium and provides sufficient substrate reserves for bgaB in the signal module.

The Bacillus thermoproteolyticus-derived bgaB inherently exhibits excellent catalytic activity and thermal stability. Furthermore, the dry experiment-optimized bgaB can effectively adapt to the intestinal microenvironment - acidic pH, mitigating the attenuation of wild-type bgaB catalytic activity under intestinal conditions and ensuring efficient substrate-catalysis coupling.

Ultimately, via the mechanism "intestinal lactose → LacY mediated substrate import → BgaB catalyzed substrate conversion", the target system enables lactose-dependent targeted responses and efficient function execution. This not only addresses the innate limitation of insufficient lactose uptake in Bifidobacterium but also enhances the system's intestinal adaptability through dry experiment-based bgaB optimization, representing a core prerequisite for the engineered strain to realize intestinal therapeutic functions via lactose metabolism.

Form

The primary objective is to enhance the expression of LacY to increase the number of lactose transporter proteins on the membrane and promote the expression of BgaB to accelerate substrate decomposition, thereby reducing the concentration of lactose in the environment.

Our adhesion system consists of two main parts:

LacY (lactose permease) --- enhances lactose absorption capacity in the gut of engineered bacteria

BgaB (β-galactosidase) --- enables prompt and efficient degradation of absorbed lactose

Module2- Check

We purified the target protein via affinity chromatography using a GST tag, then quantitatively assessed its catalytic activity through a colorimetric assay. The core principle involves the colourless substrate ONPG (o-nitrophenyl-β-D-galactopyranoside) undergoing specific hydrolysis by lactase, yielding the yellow product o-nitrophenol (ONP). By detecting changes in absorbance at 420 nm using a spectrophotometer and constructing an enzyme activity time curve, the enzymatic reaction rate can be quantified. Beyond in vitro enzyme assays, we have established an ONPG liquid detection method for rapid evaluation of lactase function in Bifidobacterium longum transformants. This technique involves co-culturing engineered bacteria with ONPG; successful expression and transport of lactase by the bacteria will hydrolyse intracellular ONPG, producing a yellow product that causes the bacterial culture to develop colour. This provides indirect evidence of lactose partial system functionality.

Result

The expression of the target gene in the engineered bacteria

The target gene was amplified via PCR and subjected to homologous recombination with the plasmid PMB1-A isolated from Bifidobacterium longum strain B2577, which had been digested by EcoR I and Xba I enzymes. The ligation product was then introduced into competent engineered bacteria for plate cultivation. After 12-16 hours, well-defined single colonies were selected for colony PCR analysis, followed by gel electrophoresis to verify the successful gene transfection. The experimental procedures including target gene amplification, enzyme digestion, plasmid recovery, and PCR gel electrophoresis are detailed below:

After completing the colony PCR and eliminating the false positive bacteria, we selected a single positive colony for the sequence detection, and successfully obtained the engineered bacteria containing pGAP+LacY respectively.

Lactase activity assay following modification

Troubleshooting and cause analysis for initial protein purification failure

Protein Not Detected Due to Promoter Species Compatibility Issues

The initial purification was based on the assumption that the target promoter was a constitutive promoter. No induction treatment was applied; instead, engineered bacteria were directly expanded in culture. Protein distribution across purification steps was monitored via SDS-PAGE (samples analysed included: logarithmic phase culture, ultrasonic lysate pellet, Wash buffer eluate, and the first 3 mL of Elution buffer eluate). Ultimately, no expected target bands were observed in any sample.

Subsequent literature review identified a critical issue: the promoter's 'constitutive expression characteristics are only compatible with yeast systems'. In Escherichia coli, glycerol or glucose must be added as 'protein stability regulators' to maintain expression levels. Supplementary experiments were conducted accordingly: cultures were expanded in LB medium supplemented with either 2% glycerol or 6% glucose, followed by repetition of the purification and SDS-PAGE analysis protocols. The target protein remained undetectable. This preliminary result rules out 'protein degradation due to stabiliser deficiency' as the sole factor, suggesting additional compatibility issues may be present.

Multivariate optimisation achieves successful target protein expression

Addressing initial failures, pilot-scale experiments focused on three core variables------'strain selection, induction strategy, and temperature control'------ultimately achieving effective target protein expression. The specific optimisation pathways and outcomes are as follows:

① Strain optimisation: Selected BL21 (DE3), a dedicated prokaryotic protein expression strain

Drawing upon the established practice in literature that 'E. coli protein purification prioritises the BL21 (DE3) strain' (which contains the T7 RNA polymerase gene, is compatible with most prokaryotic expression vectors, and its protease-deficient background reduces target protein degradation), the original strain was replaced, laying the foundation for subsequent induction.

② Induction Strategy Adjustment: Prokaryotic vs. Eukaryotic Expression Differences

Considering that 'promoters may not be strictly constitutive in E. coli' and the fundamental differences in expression regulation mechanisms between prokaryotes and yeast (eukaryotes), IPTG induction was attempted (final concentration 0.5 mmol/L). SDS-PAGE analysis revealed a distinct 32.8 kDa band in the lysate supernatant following induction, consistent with the target protein's molecular weight.

③ Induction Temperature Optimisation: Balancing Protein Folding Efficiency and Band Quality

To prevent excessive folding at high temperatures leading to inclusion body formation, three induction temperature gradients were established at 16°C, 20°C, and 37°C (all at 160 rpm for 19 hours induction). Band characteristics were compared via SDS-PAGE:

37°C group: Target band present with highest intensity and most uniform width

16°C group: Band intensity dimmer, lane background slightly blurred

20°C group: Target band intensity brighter

The optimal conditions for subsequent protein extraction were ultimately selected as '20°C, 160 rpm, IPTG induction for 19 hours'.

Based on the results from our previous tests, we optimized the bacterial culture conditions for this experiment. The final induction parameters were set as follows: IPTG induction at 20°C with shaking at 160 rpm for 16 hours.

Due to a malfunction of the high-pressure homogenizer, cell lysis was temporarily performed using an ultrasonic disruptor. Compared to the homogeneous physical shearing and efficient wall-breaking achieved by high-pressure homogenization, the localized energy concentration in ultrasonic disruption may lead to two potential issues:

① structural denaturation of some target proteins due to localized heating or mechanical forces, and

② incomplete cell lysis, leaving a portion of the target protein trapped within unbroken cells, thereby indirectly reducing subsequent extraction efficiency.

However, it is noteworthy that positive signals were observed during the experiment: bands suspected to be the target protein were detected in samples from the induction stage, and similar signals were also found in the wash buffer from the binding resin step. This indicates that the target protein was most likely expressed successfully after induction. The fact that it was not ultimately obtained in the elution fraction suggests that the core issue likely lies in the fusion between the target protein and its affinity tag --- specifically, if the tag was not successfully conjugated or presented, it would prevent effective binding to the purification resin.

Reflections on Exploratory Experiments for Bifidobacterium longum Electroporation Conditions

1. First electrical transfer test

① Competent Cell Preparation:

• Culture B. longum to mid-logarithmic growth phase.

• Critical Step 1 (Cell Washing): Wash cells using distilled water.

• Critical Step 2 (Cell Resuspension): Use Electroporation Buffer: 1 mM citrate buffer (2.52 mg citric acid, 22.42 mg trisodium citrate, 1 L distilled water, pH 6.0) containing 0.5 M sucrose, pH 6.0.

② Electrotransformation Parameters:

•Use the pre-programmed Pichia mode (Pic) on the capacitance discharge electroporator (resulting in approximately 5 ms pulse duration).

③ Post-Electroporation Treatment:

• Immediately after pulsing, add 1 mL of pre-warmed (37°C) MRS liquid recovery medium containing 2% sucrose.

• After anaerobic recovery, spread onto MRS selective plates containing 20 μg/ml ampicillin.

④ Results:

• Antibiotic plates: No single colonies appeared.

• Negative control: No colonies observed.

⑤ Critical Cause Analysis for the First Attempt Failure:

Fatal Flaw of Distilled Water Washing: Using hypotonic distilled water for washing causes a massive influx of water into the bacterial cells, leading to cell swelling and potential rupture, resulting in irreversible damage and massive cell death. Even if some cells survive, their physiological state is severely compromised, making them unable to withstand subsequent electroshock and recovery steps. This is the most probable primary reason for the failure of the first experiment.

2. Second Electrotransformation Attempt

① Competent Cell Preparation:

• Culture B. longum to mid-logarithmic growth phase.

• Critical Step (Cell Washing): Replace distilled water washing with an isotonic 10% glycerol solution containing 2% sucrose. Use the same isotonic buffer for all washing and resuspension steps.

• The Electroporation Buffer remains unchanged.

② Electrotransformation Parameters:

• Fixed pulse duration: 1 ms. Scanned voltage: 1.8 kV.

③ Post-Electroporation Treatment:

• Identical to the first attempt.

④ Results:

• Antibiotic plates: No single colonies appeared.

• Negative control: No colonies observed.

⑤ Rationale and Value of the Second Attempt Improvement:

Improvement Reason: To address the issue of hypotonic shock encountered in the first experiment. Using an isotonic buffer containing 2% sucrose and 10% glycerol aims to provide a constant and stable environment for the cells throughout the washing and resuspension process, maximizing cell integrity and viability, and ensuring that the starting cell population used for electroporation is healthy and consistent.

Reflection: Although this modification still did not yield success, it successfully eliminated the key confounding factor of "osmotic shock causing massive cell death," thereby shifting the focus more clearly onto the electroporation parameters themselves.

3. Conditional exploration trial

First Electrotransformation Condition Screening Experiment

① Competent Cell Preparation:

• As described above (using improved washing buffer).

② Electrotransformation Parameters:

• Fixed pulse duration: 4 ms.

• Tested voltage gradient:

1.8 kV, 4 ms (Sample 1)

1.9 kV, 4 ms (Sample 2)

2.0 kV, 4 ms (Sample 3)

2.1 kV, 4 ms (Sample 4)

2.2 kV, 4 ms (Sample 5)

2.3 kV, 4 ms (Sample 6)

2.4 kV, 4 ms (Sample 7)

2.5 kV, 4 ms (Sample 8)

• Control: 1.8 kV, 4 ms (with ddH₂O instead of DNA, blank)

③ Post-Electroporation Treatment:

• Identical to the first attempt (recovery in 37°C MRS + 2% sucrose, then plating).

④ Results: No single colonies appeared on any antibiotic plates across the entire voltage gradient.

⑤ Rationale and Value of the First Screening Experiment:

Systematic Parameter Exploration: After addressing major issues in cell preparation, this experiment began systematically scanning combinations of voltage and time, marking a shift from "eliminating critical errors" to "searching for optimal conditions."

Second Electrotransformation Condition Screening Experiment

① Competent Cell Preparation:

• As described above (using improved washing buffer).

② Electrotransformation Parameters:

• Fixed pulse duration: 3 ms.

• Tested voltage gradient:

1.8 kV, 3 ms (Sample 1)

1.9 kV, 3 ms (Sample 2)

2.0 kV, 3 ms (Sample 3)

2.1 kV, 3 ms (Sample 4)

2.2 kV, 3 ms (Sample 5)

2.3 kV, 3 ms (Sample 6)

2.4 kV, 3 ms (Sample 7)

2.5 kV, 3 ms (Sample 8)

• Control: 1.8 kV, 3 ms (with ddH₂O instead of DNA, blank)

③ Post-Electroporation Treatment: Identical to the first screening experiment.

④ Results: Still no single colonies appeared on any MRS antibiotic plates across the voltage gradient.

⑤ Reflection on the Second Screening Experiment:

Given the relative immaturity of the B. longum electrotransformation protocol, we suspected potential cell death during competent cell preparation or the electroporation step itself. To test this, we used the same MRS solid medium without ampicillin. Both the prepared competent cells and the post-electroporation cells from each group were recovered, centrifuged, and the concentrated cell suspensions were plated onto both antibiotic-containing and antibiotic-free plates to observe the results.

Third Electrotransformation Condition Screening Experiment

① Competent Cell Preparation:

• Continued using the improved method from the second attempt (washing with 10% glycerol + 2% sucrose buffer).

• Modified Electroporation Buffer: Changed to 0.5 mM MgCl₂ - 0.5 M sucrose buffer.

② Electrotransformation Parameters:

• Core Variables: Systematically varied both voltage and pulse time.

• Specific Condition Combinations:

2.5 kV, 4 ms (Sample 1)

2.4 kV, 4 ms (Sample 2)

2.3 kV, 4 ms (Sample 3)

2.6 kV, 4 ms (Sample 4)

2.6 kV, 3 ms (Sample 5)

2.5 kV, 3 ms (Sample 6)

2.5 kV, 2 ms (Sample 7)

2.6 kV, 2 ms (Sample 8)

③ Post-Electroporation Treatment:

• Key Improvement: Divided the recovered culture from each condition into two parts, plating onto MRS plates with and without 20 μg/ml ampicillin.

• Included Negative Control: Plated non-electroporated competent cells onto non-selective (no AMP) plates.

④ Results:

• Antibiotic Plates: No single colonies.

• Non-selective Plates: All conditions showed dense growth of single colonies, demonstrating that B. longum cells remained viable after the electroporation procedure.

• Negative Control: Showed dense growth of single colonies, confirming that the competent cells were viable after the preparation process.

⑤ Reflection on the Third Screening Experiment:

The results demonstrated that the bacterial cells remained viable both after the centrifugation steps during competent cell preparation and after the electroporation pulse itself. However, no transformants were obtained on the selective plates.

The essence of electroporation is to apply a sufficiently high and appropriately long transmembrane voltage to induce the formation of temporary, reversible hydrophilic pores in the phospholipid bilayer, allowing foreign DNA to enter.

The core function of the electroporation buffer in this process is to regulate the overall resistance of the system. This resistance directly determines whether a specific applied voltage can generate a sufficiently strong electric field strength and maintain it for an adequate duration.

Having adjusted the voltage conditions to the optimal electric field strength of 12.5 kV/cm based on literature, we suspected that the pulse duration and the ionic composition/ratio of the electroporation buffer might be the most critical limiting factors for success. However, given that the maximum pulse duration achievable with our current electroporator was 4.0 ms, we decided to attempt to compensate for this limitation by further increasing the voltage and by comparing the outcomes using two different buffer formulations.

Fourth Electrotransformation Condition Screening Experiment

① Competent Cell Preparation:

• Maintained the improved washing steps.

• Introduced a new core variable: Two different Electroporation Buffers.

Buffer A: Citric acid - Sodium citrate - Sucrose buffer, pH 6.0

Buffer B: 0.5 mM MgCl₂ - 0.5 M Sucrose buffer

② Electrotransformation Parameters:

• Core Variable: Tested different buffers at higher voltages.

• Specific Condition Combinations (applied for each buffer):

2.7 kV, 4 ms, Buffer B (Sample 1)

2.8 kV, 4 ms, Buffer B (Sample 2)

2.275 kV, 4 ms, Buffer B (Sample 3)

2.65 kV, 4 ms, Buffer B (Sample 4)

No pulse, Buffer B (Blank 1)

2.7 kV, 4 ms, Buffer A (Sample 5)

2.8 kV, 4 ms, Buffer A (Sample 6)

2.275 kV, 4 ms, Buffer A (Sample 7)

2.65 kV, 4 ms, Buffer A (Sample 8)

No pulse, Buffer A (Blank 2)

③ Post-Electroporation Treatment: Identical to the third experiment (plating on both AMP-containing and non-selective plates, including negative controls).

④ Results:

• Resistant plates: No single colonies observed.

•Non-resistant plates: Dense single colonies emerged on plates 1, 3, and 4. This demonstrates that the 0.5 mM magnesium chloride-0.5 M sucrose buffer solution effectively enhances the survival rate of Bifidobacterium longum following electroporation.

• Negative control: Both blanks 1 and 2 yielded dense single colonies. This demonstrates that Bifidobacterium longum remained viable following preparation of the competent cells.

⑤ Reflection on the fourth Screening Experiment:

Electroporation parameters (voltage/duration) at 2.75--2.8 kV and 4 ms yielded reversible damage to the cell membrane, with cells capable of repair and recovery. This excludes the possibility that 'electroporation itself directly killed all cells'. Cell viability under buffer B conditions was significantly higher than under buffer A (the latter yielding no colonies on the agar plate). This indicates that Mg²⁺ ions play a crucial role in protecting cells, maintaining cell membrane stability, or promoting membrane repair. Mg²⁺ serves as a cofactor for numerous enzyme systems and may facilitate membrane repair post-electroporation. We therefore hypothesise that either the exogenous plasmid DNA failed to successfully enter the cells, or that it did not successfully stabilise, maintain, and express itself within the cells.

Based on the aforementioned experimental data, subsequent trials will employ new gradient voltage electroporation conditions at 2.75 kV and 2.8 kV, with pulse durations of 3--4 ms, to determine the optimal electroporation parameters for Lactobacillus longum ATCC 15707. (Equipment limitations restrict the maximum electroporation pulse duration to 4 ms.)

Dual-Plasmid Co-Detection in E. coli

Given the inherent difficulty of transforming Bifidobacterium longum, we employed an alternative approach using E. coli, which possesses fewer lactose transporters on its cell membrane, for a preliminary dual-plasmid assay. This system co-expressed the lactose-degrading enzyme and a lactose pump. Simultaneous expression of both proteins was confirmed by monitoring absorbance changes at 420 nm and 500 nm using a spectrophotometer. The enzymatic activity, particularly the baseline activity of the engineered lactase, was qualitatively assessed by calculating the average absorbance values and applying established calculation methods from the literature.

(1) Enzyme Activity Assay

The enzymatic activity was analyzed using the ONPG-ONP method. Absorbance was measured at 420 nm, specific for ONP, and at 500 nm for turbidity correction. The activity was calculated using the formula:

a_sp = (net OD₄₂₀ × V_t) / (ε × l × t × V_t)

the derivation from the Lambert-Beer Law：

ε = A / (c × l)

Defines the physical quantities: ε is the molar extinction coefficient of the product (ONP), c is its molar concentration, l is the path length of the cuvette, and A is the absorbance at a given time.

(2) Qualitative Screening and Result Interpretation

Qualitative visual inspection of 30 test groups was conducted. Based on the expected plasmid transformation efficiency, groups exhibiting the most pronounced color change were prioritized as the most likely hosts for successful dual-plasmid co-transformation. After blank correction, the average difference between OD₄₂₀ and OD₅₀₀ measured at 25 minutes was 0.0149. This result indicates a certain level of lactase activity. Given that E. coli itself has very poor lactose uptake and metabolism, it is reasonable to conclude that the observed lactase activity primarily originated from the heterologously expressed lactose metabolic system. This confirms that the fundamental metabolic function of our engineered lactase was preserved in the dry lab phase.

(3) Conclusion and Follow-up Strategy

However, it is important to note that the constructed shuttle plasmids (for E. coli and Bifidobacterium) impose a significant expression burden on the host. This burden, exacerbated by the presence of other exogenous genes on the same plasmid, substantially impacted the measured enzyme activity in this in vivo assay. Therefore, we propose to proceed with the purification of the lactase followed by in vitro activity assays for more accurate quantification.

Image: GST (media/image20.png)

Reference

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Module1- Design

Engineering Bacteria Selection: "Model Verification" to "Application Implementation" Plasmid Compatibility Selection

Function realization

We selected deoxycholic acid (DCA) as the upstream targeted regulatory signal for the intestinal adhesion-colonization system, with the transcriptional repressor BreR serving as the core regulatory component[1]. Notably, previous literature has reported that BreR exhibits specific responsiveness to DCA[2] - a key characteristic that supports its role in mediating DCA-dependent regulation of the adhesion-colonization system. At the molecular level, BreR specifically binds to the transcription factor-binding site (TFBS) region of promoter pGAP, inhibiting downstream functional genes (in this system, MucBP, an adhesion-related protein-coding gene) through steric hindrance[3][4]. In the human digestive tract, the colon serves as the final destination for lactose consumed by lactose-intolerant patients - where various bacteria break down lactose, producing large amounts of insoluble gases like hydrogen. These gases rise upward, causing intestinal gas that disrupts patients' daily lives. Compared to other digestive regions, colon DCA levels are significantly higher than in the stomach and small intestine[5]. This localized high concentration of DCA enables efficient and specific binding to BreR, inducing conformational changes that detach BreR from the pGAP(TFBS) site[6] - This process rapidly reverses transcriptional repression on the MucBP gene, triggering its efficient expression. Through a series of experiments, we achieved specific adhesion and colonization of engineered bacteria containing the target system in the colon, effectively avoiding non-targeted expression in other digestive regions.

Form

Our experimental plan is divided into three parts: the connection and expression of deoxycholic acid-responsive gene BreR with high expression promoter pGAP (TFBS) and mucin-binding protein gene MucBP with high expression promoter breAB in the engineered bacteria, as well as the verification of these two connections and the expression of the successfully sequenced plasmid in the engineered bacteria.

Our adhesion system consists of three main parts:

The input signal — High concentration of deoxycholic acid triggers the adhesion system

Signal pathway — BreR responds to deoxycholic acid and relieves transcriptional repression to facilitate the colonization of engineered bacteria in the colon

The output signal — Modified engineering bacteria were colonized in the colon

Module2 - Check

We plan to use two approaches to test the adhesion system.

In the first approach, we plan to use the Caco-2 cell line derived from human colon cancer cells as the substrate. By co-culturing the modified engineered bacteria with Caco-2 cells for 1-2 hours and measuring the fluorescence intensity, we will compare the results with those obtained when the unmodified engineered bacteria are cultured together. This comparison aims to validate the anticipated conclusion that the modified Escherichia coli DH5α demonstrates significantly stronger adhesion capability than its unmodified counterpart.

The Caco-2 cell line, derived from human colon cancer cells, can differentiate into a single-layered intestinal epithelial cell membrane under in vitro culture conditions, making it an ideal substrate for cellular colonization [7]. Fluorescence-labeled co-culture, as a well-established detection method, offers advantages such as clear results and rapid experimentation [8], thus being selected as the primary experimental protocol.

For the second approach, we developed a method using qualitatively treated filter paper with porcine gastric mucosal proteins as the substrate. After inoculating the bacterial suspension into sterile petri dishes with LB medium petri dishes at 37°C for 1-2 hours under aerobic conditions, the cultures were washed and spread across the solid medium surfaces in sterilized petri dishes. The modified engineered bacteria were incubated for 48-72 hours until colonies became clearly visible. Comparative analysis of colony counts between modified and unmodified strains confirmed that the engineered bacteria exhibited significantly enhanced adhesion capabilities compared to their original counterparts.

As a mucin expression gene, MucBP can colonize a specific location by binding to mucosal proteins. The growth of colonies also directly reflects adhesion ability of the bacterial strains. Therefore, this experiment was selected as the second experimental plan due to its simple operation and concise experimental steps. [4]

Result

Through our efforts, we have achieved a series of results to validate our hypothesis. In the adhesion system section, we successfully developed engineered bacteria expressing BreR and MucBP genes separately, and conducted attempts to co-express both genes within a single Escherichia coli colony. Due to unfavorable experimental conditions in chemical transformation, we have not yet obtained a single colony containing both genes. However, we will continue optimizing the protocol in subsequent experiments to achieve success as soon as possible. Our experimental results and procedures are detailed below, providing insights into our research achievements.

The expression of the target gene in the engineered bacteria

The target gene was amplified via PCR and subjected to homologous recombination with the plasmid PMB1-A isolated from Bifidobacterium longum strain B2577, which had been digested by EcoR I and Xba I enzymes. The ligation product was then introduced into competent engineered bacteria for plate cultivation. After 12-16 hours, well-defined single colonies were selected for colony PCR analysis, followed by gel electrophoresis to verify the successful gene transfection. The experimental procedures including target gene amplification, enzyme digestion, plasmid recovery, and PCR gel electrophoresis are detailed below:

After completing the colony PCR and eliminating the false positive bacteria, we selected a single positive colony for the sequence detection, and successfully obtained the engineered bacteria containing pGAP+BreR and pGAP+MucBP respectively.

After receiving the two kinds of genetically engineered bacteria respectively, we carried out revival culture and transformation of the BreR gene engineered bacteria.

Expression of two plasmids in engineered bacteria

We chose to use Ca+ transformation method to chemically transform the engineered bacteria which were confirmed to contain the BreR gene with correct sequencing, and then introduce the plasmid containing MucBP gene into them to obtain the engineered bacteria containing both genes. However, some problems were encountered in this process.

During the first transformation, many microcolonies that are difficult to isolate grew on ourresistance plate, which we speculated were caused by the high concentration of calcium ions duringthe experiment". Therefore, we changed the 0.1 M/L CaCl2 used in the transformation to a mixture of0.08 M/L MgCl2 and 0.02 M/L CaCl2 and conducted the experiment again.

During the second chemical transformation, our bacterial colony number was too large to form a bacterial film covering the whole surface of the culture medium and single bacterial colonies could not be separated. After literature search and discussion, we believed that the high concentration of plasmid during transformation was the cause.

In response to this phenomenon, we controlled the amount of transferred plasmid during the second transformation and reduced it to 40% of the first transformation, but the result was still the formation of bacterial membrane. The control group also formed bacterial membrane, which proved that the main problem lies in the engineered bacteria used for preparing the receptive state.

Through further investigation, we concluded that the root cause likely lies in the resistance genes used by both MucBP and BreR genes being ampicillin resistance genes. During chemical transformation, engineered bacteria can be categorized into two groups: those without MucBP transfection and those with MucBP transfection. In reverse transcription culture, since the untransfected bacteria naturally possess resistance genes, their screening efficiency on ampicillin-resistant plates is significantly reduced. This results in both transfected and non-transfected engineered bacteria proliferating extensively, ultimately covering the entire surface of the resistance plate.

Follow-up experiments

If we successfully construct an engineered bacterium containing both genes, we will test it using two methods we previously proposed: the Caco-2 cell assay and filter paper coated with mucosal protein assay. Here is our protocol: Adhesion System Protocol

References

[1] Xu, J., Zhang, J., Wang, X., Hao, Y., Wang, J., Su, B., Yin, J., Wang, Z., Wang, Q., & Qi, Q.[1] Xu, J., Zhang, J., Wang, X., Hao, Y., Wang, J., Su, B., Yin, J., Wang, Z., Wang, Q., & Qi, Q. (2025). Directed Evolution of Transcriptional Factor BreR for Improved Substrate Specificity Toward Deoxycholic Acid and Ursodeoxycholic Acid. Biotechnology Journal, 20(6). [2] Cerda-Maira FA, RingeLBerg CS, Taylor RK. The bile response repressor BreR regulates expression of the Vibrio cholerae breAB efflux system operon. J Bacteriol. 2008;190(22):7441-7452. [3] Devi, S. M., & Halami, P. M. [3] Devi, S. M., & Halami, P. M. (2017). Diversity and evolutionary aspects of mucin binding (MucBP) domain repeats among Lactobacillus plantarum group strains through comparative genetic analysis. Systematic and Applied Microbiology,40(4), 237–244. [4] Chatterjee M, Pushkaran AC, Vasudevan AK, Menon KKN, Biswas R, Mohan CG. Understanding the adhesion mechanism of a mucin binding domain from Lactobacillus fermentum and its role in enteropathogen exclusion. Int J Biol Macromol. 2018;110:598-607. [5] Jingjing Cong, Pianpian Liu, Zili Han, Wei Ying, Chaoliang Li, Yifei Yang, Shuling Wang, Jianbo Yang, Fei Cao, Juntao Shen, Yu Zeng, Yu Bai, Congzhao Zhou, Lilin Ye, Rongbin Zhou, Chunjun Guo, Chunlei Cang, Dennis L. Kasper, Xinyang Song, Lei Dai, Linfeng Sun, Wen Pan, Shu Zhu, Bile acids modified by the intestinal microbiota promote colorectal cancer growth by suppressing CD8+ Tcell effector functions, Immunity, Volume 57, Issue 4,2024, Pages 876-889.e11, ISSN 1074-7613 [6] Beabout K, Ehrenworth Breedon AM, Blum SM, [6] Beabout K, Ehrenworth Breedon AM, Blum SM, et al. Detection of Bile Acids in Complex Matrices Using a Transcription Factor-Based Biosensor. ACS Biomaterials Science & Engineering. 2022;9(9):5151-5162. [7] Isidro-Coxca MI, Ortiz-Jiménez S, Puente JL. Type 1 fimbriae and P pili: regulatory mechanisms of the prototypical members of the chaperone-usher fimbrial family.Arch Microbiol. 2024;206(9):373. Published 2024 Aug 10. [8] Riedel CU, Foata F, Goldstein DR, Blum S, Eikmanns BJ. Interaction of bifidobacteria with Caco-2 cells-adhesion and impact on expression profiles. Int J Food Microbiol. 2006;110(1):62-68.

Module1- Design

Engineering Bacteria Selection: "Model Verification" to "Application Implementation" Plasmid Compatibility Selection

Function realization

This study utilizes AI-2 as the core sensing signal for gut microbiota density. The core regulatory element employs a dual-gene combination of "LuxS signaling gene and CcdB proliferation inhibitor"[2]. Its density-responsive mechanism dependent on AI-2 has been validated through gut microbiota research, establishing a critical molecular foundation for achieving dynamic equilibrium in intestinal flora.

When AI-2 levels fall below the response threshold, LuxS gene-coding S-nucleosyl homocysteine lyase continuously catalyzes the synthesis of DPD (4,5-dihydroxy-2,3-pentanediol). The spontaneous rearrangement of DPD into AI-2 forms an "endogenous signaling reservoir". Simultaneously, exogenous AI-2 produced by gut microbiota (e.g., Escherichia coli and Bifidobacterium) synergistically accumulates with endogenous AI-2 to form a "density-responsive signal source" [3]. The CcdB gene (DNA gyrase inhibitory toxin CcdB) is positioned downstream of the AI-2 concentration-responsive promoter (breAB) [1]. When AI-2 concentrations remain below the promoter activation threshold, CcdB expression is suppressed, allowing the engineered bacteria to proliferate normally for intestinal colonization. However, when the microbial density in the gut becomes excessively high (containing both engineered bacteria and natural flora), the combined concentration of AI-2 (endogenous + exogenous) reaches the response threshold. This elevated AI-2 acts as a molecular switch, specifically binding to the promoter's regulatory protein to trigger efficient CcdB expression. By inhibiting key bacterial DNA replication enzymes, CcdB rapidly restricts excessive proliferation of both engineered bacteria and surrounding microbial populations, preventing ecological disruption caused by overcrowding.

Ultimately, through the mechanism of "intestinal microbiota → AI-2 concentration gradient → LuxS signal perception + CcdB response regulation → dynamic balance of microbial density", the system achieves precise sensing and autonomous regulation of gut microbiota density. This effectively prevents intestinal dysfunction caused by over-implantation of engineered bacteria or imbalance in natural flora, thereby ensuring the safe application and ecological compatibility of gut engineering bacteria.

Form

Input signal: AI-2 signaling molecule, catalyzed by LuxS enzyme, accumulates with the increase of bacterial density.

Signal pathway: The AI-2 concentration-responsive promoter-pslrA activates the downstream gene CcdB expression.

Output response: CcdB virulence protein expression induces the death of some cells in high-density bacterial flora to achieve population density regulation.

Module2- Check

This experiment aims to construct an engineered bacterial regulation module based on LuxS/AI-2 quorum sensing system[5], using AI-2 signaling molecule as the input of quorum density perception, and realizing negative feedback regulation of bacterial density through the expression of CcdB virulence protein.

LuxS protein, a key enzyme in AI-2 signaling molecule synthesis, is widely distributed across various bacterial species. As a universal quorum-sensing signal, AI-2 can be recognized and regulated by different bacterial strains to coordinate collective behavior[6][7]. We selected Vibrio harveyi BB170 as the reporter strain for AI-2 activity detection. That’s because of this strain lacks the LuxS gene and cannot synthesize AI-2, but it retains a complete sensing and signaling system. The LuxP/LuxU/LuxO pathway enables the bacteria to detect exogenous AI-2 and activate fluorescent expression, with luminescence intensity showing a positive correlation with AI-2 concentration.

The DPD rearranges to form AI-2, and the promoter with the expression of the downstream LuxS gene can help the engineering bacteria produce DPD.

Secondly, CcdB protein functions as a DNA gyrase toxin that induces cell death by disrupting the DNA replication process. By inserting the CcdB gene downstream of the AI-2-responsive promoter, we established an AI-2 concentration-dependent expression system. As bacterial population density increases, the AI-2 concentration rises accordingly, activating downstream CcdB expression. This mechanism enables self-cleaning of high-density bacterial colonies and maintains microbial homeostasis.

Result

Construction of the plasmid

The analysis of the electrophoresis results of the plasmids we constructed showed that both plasmids were successfully constructed, as shown in the figure:

Detection of CcdB toxicity

We provide different concentrations of DPD (4,5-dihydroxy-2,3-pentanediol) which can be rearranged to generate AI-2 and the concentration is proportional to AI-2 and we can analyze the inhibitory effect of different DPD concentrations on the growth curve of engineering bacteria by detecting OD600

Compared with the engineering bacteria control group without adding exogenous AI-2 in Figure 3.1, the experimental groups with different concentrations of exogenous AI-2 showed a certain inhibitory trend on the growth of engineering bacteria, but the inhibitory effect did not reach a significant level and did not show obvious concentration-dependent inhibitory law

Due to time constraints preventing timely acquisition of specific concentration AI-2 reagents required for subsequent experiments, it became challenging to continue optimizing the concentration gradient of exogenous AI-2. More crucially, the core objective of this study was to establish a closed-loop system —— where engineered bacteria autonomously produce AI-2 through suicide regulation. This would utilize the bacteria's self-synthesized AI-2 as a signaling mechanism to activate the toxicity system, rather than relying on external supplementation. Consequently, we revised our experimental strategy by abandoning further exploration of exogenous AI-2 and instead designing an endogenous AI-2-toxicity system integrated experiment. This approach better aligns with the density-dependent regulation requirements in practical application scenarios.

Detection of AI-2 activity

After filtration, the LB and AB media were used for cultivation. The engineered bacteria were cultured in LB medium, while BB170 was cultivated in AB medium. Sterile supernatants from each strain were collected (with the culture medium serving as a control, filtered, sterilized, and cryopreserved). BB170 cells activated to appropriate density were diluted in AB medium and mixed with the respective supernatants in proportion for co-cultivation. Within 0-6 hours, luminescence intensity in the 96-well plate system was measured every 30 minutes using a chemiluminescent model from an enzyme-linked immunoassay (EIA) instrument. Relative fluorescence intensity was calculated and compared with positive controls to determine AI-2 activity.

Figure 3.2: It shows that the supernatant of the tested bacteria contains AI-2 signaling molecules, so that the concentration of AI-2 signaling molecules in the mixed solution is greater than that in the negative control group, so that the concentration of inducing indicator bacteria to glow can be achieved earlier, and the time of increasing fluorescence intensity is earlier than that in the negative control.

As shown in Figure 3.2, within the first 4 hours, fluorescence intensity values in all experimental groups (except the positive control) decreased over time. The positive control group exhibited a gradual decline in fluorescence intensity during the initial 0~3.5h hours, reaching its lowest point at 3.5 hours. Subsequently, fluorescence intensity began to rise with extended observation time. Therefore, we selected 4 hours as the baseline for subsequent calculations.

The activity of AI-2 is expressed by relative fluorescence intensity, and the calculation formula is as follows[4]:

As shown in Figure 3.3, AI-2, as a quorum sensing signaling molecule, is highly correlated with bacterial growth density - reaching its peak activity from the late logarithmic phase to the early stationary phase (9h), which conforms to the logic of "no synthesis at low density and massive release at high density" of quorum sensing.

Joint detection

Through individual testing of each component, we observed that the LuxS component can produce active AI-2 in engineered bacteria, while the CcdB component also demonstrates bactericidal activity. However, since the concentration and activity of LuxS's AI-2 cannot be directly correlated with the DPD introduced by our CcdB system, and given time constraints, we opted to simultaneously introduce both plasmids into the engineering bacteria and conduct single colony count measurements at different time points.

TThe results are as follows:

The PCR bands after the transfer of plasmid are shown in the figure above, and it can be seen that No.9 is engineering bacteria.

Growth trend differences: Under identical cultivation conditions, the wild-type strain maintained a steady increase in viable colony counts. However, the engineering bacteria No.4 and No.9 containing dual plasmids showed a marked decline in single colony counts over time —— directly demonstrating that co-expression of LuxS and CcdB systems inhibits bacterial growth in engineered strains.

After 2-hour cultivation, the viable colony counts of Project Bacteria No.9 peaked (indicating dense bacterial distribution), followed by a rapid decline. This phenomenon perfectly aligns with the "dual-system density-dependent regulation hypothesis" - when bacterial density becomes excessive, the AI-2 produced by LuxS accumulates to a threshold level, triggering the CcdB system to activate its antimicrobial function and suppress proliferation. Therefore, when density exceeds a critical threshold, the CcdB-mediated action can suppress proliferation, preventing overgrowth and ultimately achieving dynamic equilibrium in microbial density.

However, under identical incubation conditions, the viable count of wild-type strains with empty plasmids was significantly lower than that of the engineering bacteria No.9 containing LuxS-CcdB dual plasmids. This contradicts the initial hypothesis that "WT strains without lethal systems should exhibit normal growth and higher population numbers." Analyzing experimental design and operational details, potential causes can be attributed to four key factors: initial inoculation variations, strain genetic background differences, inconsistent cultivation conditions, and procedural errors. While "inconsistent bacterial inoculation quantities" constitutes a direct contributing factor, it is not the sole determinant.

Of course, the consistent bacterial inoculation volume should be taken into account. In subsequent experiments, we will adjust the protocol by adjusting the test bacterial suspension, engineered bacteria strains No.9, and WT to achieve identical OD600 values (e.g., OD600=0.1, corresponding to approximately 10⁷ CFU/mL for Escherichia coli) using sterile LB medium after recovery. This ensures complete consistency in initial inoculation concentration before proceeding with subsequent experiments.

Follow-up experiments

Subsequent experiments will further optimize the alignment between LuxS promoter strength and CcdB expression windows to enhance system response sensitivity and specificity. We also plan to introduce the system into Bifidobacterium longum to validate its applicability in intestinal probiotics. Additionally, we aim to develop an AI-2 concentration gradient-regulated CcdB expression model to achieve more precise microbial density control.

Here's our protocol:

protocol.docx

Vibrio harveyi BB170 report strain tested for AI-2 activity-protocol.docx

CcdB toxicity verification-protocol.docx

Verify the correlation between CcdB and AL-2 systems-protocol.docx

Reference

[1]Li JAttila C, Wang LWood TK, Valdes JJ, Bentley WE2007.Quorum Sensing in Escherichia coli Is Signaled by AI-2/LsrR: Effects on Small RNA and Biofilm Architecture. J Bacteriol 189 [2]Van Melderen L. Molecular interactions of the CcdB poison with its bacterial target, the DNA gyrase. Int J Med Microbiol. 2002 Feb;291(6-7):537-44. [3]Zhu P, Li M. Recent progresses on AI-2 bacterial quorum sensing inhibitors. Curr Med Chem. 2012;19(2):174-86. [4]Surette MG, Bassler BL. Quorum sensing in Escherichia coli and Salmonella typhimurium. Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):7046-50. [5]Chen, X., Schauder, S., Potier, N. Chen, X., Schauder, S., Potier, N. et al. Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415, 545–549 (2002). [6]Xavier, K. B., & Bassler, B. L. [3]Xavier, K. B., & Bassler, B. L. (2005). Interference with AI-2-mediated bacterial cell-cell communication. Nature, 437(7059), 750–753. [7]Bassler B. L. (2002). Small talk. Cell-to-cell communication in bacteria. Cell, 109(4), 421–424.