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.

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.