Introduction
1.1 Research Background
In this project, we plan to use Escherichia coli as a substitute for Edwardsiella piscicida to construct an engineered strain that exhibits linear movement under blue light and tumbling movement under green light. We will further combine this engineered strain with a light irradiation device to achieve precise control over the movement state of Escherichia coli, thereby enhancing the invasive ability of the vaccine strain and improving the efficacy of immersion immunization.
At present, we have completed the construction of four groups of plasmids, which have been introduced into Escherichia coli BL21 to construct engineered strains. We have verified that the target proteins can be successfully expressed and initially tested their effects on the movement state of bacteria.
Section 1: Plasmid Amplification and Reconstruction
In this project, pBBR1MCS-5, pDawn, and pSR58.6 were used as vectors. The main target genes included CheA, CheY, CheZ, and sfGFP. The electrophoresis results of the main fragments and vector backbones used in this study are shown in the figure below.
Figure 1 Electrophoresis Image of the CheA, CheY and CheZ Fragments
Figure 2 Electrophoresis Image of the sfGFP Fragment
Figure 3 Electrophoresis Image of the pBBR1MCS-5 Vector Backbone
Figure 4 Electrophoresis Image of the pDawn Vector Backbone
Figure 5 Electrophoresis Image of the pSR58.6 Vector Backbone
The aforementioned fragments and vector backbones were successfully assembled into the recombinant plasmids listed in the table below using a seamless cloning method.
Table 1 Main Plasmids Used in This Experiment
Section 2: Verification of Light-responsive Pathways
Centered on constructing light-controlled E. coli BL21 engineered strains that move straight under blue light and tumble under green light, this project relies on CheA, CheY, and CheZ proteins to control bacterial movement. Western Blot and fluorescence detection are key experiments to verify protein expression and regulation efficiency, laying the foundation for subsequent movement capability studies.
Western blot directly confirms whether target proteins are expressed: Using Flag-tagged plasmids (e.g., BL21-pDawn-CheZ-Flag), it detects protein expression differences under light induction. Results show blue light significantly increases CheZ expression, confirming the "light signal → target protein expression" pathway works; weak bands in dark groups indicate minor basal expression, guiding subsequent optimizations.
Figure 6 WB Development Result Image of BL21-pDawn-CheZ-Flag
Under green light induction conditions, similar results were also observed. Taking BL21-pSR43.6r&pSR58.6-CheA-Flag as an example:
Figure 7 WB Development Result Image of BL21-pDawn-CheZ-Flag
Fluorescence detection (with sfGFP as reporter gene) quantifies expression efficiency: Experiments on BL21-pDawn-sfGFP and BL21-pSR43.6r&pSR58.6 show light boosts sfGFP expression in a time-dependent manner, confirming inducible and quantifiable light-responsive pathways.
Figure 8 Relative Fluorescence Intensity of Bacterial Culture under Blue Light Induction
Figure 9 Together, they verify the light-controlled expression system is functional, ensuring reliable attribution of subsequent movement experiment results to protein function rather than expression errors.
Section 3:
Verification 1: Macroscopic Verification via Semi-Solid Medium Culture Assay
To preliminarily verify the motility of the engineered strains, we designed a semi-solid medium culture assay. Owing to its semi-solid nature, this medium exhibits both a certain degree of fluidity and stability. Utilizing semi-solid medium to assess bacterial diffusive motility (i.e., motility test) enables more intuitive observation of phenomena, making it a fundamental and crucial identification technique in microbiology laboratories.
In our hypothesis, overexpression of the CheZ protein would dephosphorylate CheY-A, thereby inhibiting the clockwise rotation of Escherichia coli (E. coli) flagella, weakening their tumbling motility, and simultaneously promoting counterclockwise rotation to enhance their straight-line motility. The weakened tumbling ability and strengthened straight-line motility of E. coli would manifest as enhanced diffusive motility on semi-solid medium, possibly with an accelerated diffusion rate—specifically, forming larger colony areas within the same time period.
To validate whether the effect of CheZ overexpression aligns with our hypothesized outcomes under IPTG induction, we constructed the pBBR1-MCS-5-CheZ system.
Figure 10 Results of semi-solid medium culture for the pBBR1-MCS-5-CheZ system(From left to right: Plates 1 and 3 are IPTG-induced groups; Plate 2 is the uninduced control group)
From the semi-solid culture results, the colonies in the two experimental groups (IPTG-induced) were indeed larger than those in the middle control group (uninduced). This confirms that overexpression of the CheZ protein can effectively promote the straight-line motility of E. coli.
Conversely, overexpression of the CheY protein or CheA protein (a phosphokinase) would increase the production of CheY-A, thereby promoting the counterclockwise rotation of E. coli flagella, enhancing their tumbling motility, and inhibiting clockwise rotation to weaken their straight-line motility. The strengthened tumbling ability and weakened straight-line motility of E. coli would manifest as reduced diffusive motility on semi-solid medium, possibly with a slower diffusion rate—specifically, forming smaller bacterial lawn areas within the same time period. To verify whether the effects of CheY and CheA overexpression match our hypothesized outcomes under IPTG induction, we further constructed the pBBR1-MCS-5-CheY and pBBR1-MCS-5-CheA systems.
Figure 11 Results of semi-solid medium culture for the pBBR1-MCS-5-CheY system(From left to right: Plates 1 and 2 are IPTG-induced groups; Plate 3 is the uninduced control group)
Figure 12 Results of semi-solid medium culture for the pBBR1-MCS-5-CheZ system(From left to right: Plates 1 and 2 are IPTG-induced groups; Plate 3 is the uninduced control group)
From the semi-solid culture results, the colonies in the IPTG-induced groups were either the same size as or smaller than those in the uninduced control group. This partially aligns with our expected outcomes. We hypothesize that this discrepancy may stem from human operational errors during sample spotting, which caused slight variations in the volume of bacterial culture added—leading to differences in colony size. To a certain extent, this result supports the validity of our initial hypothesis, but further verification is required to confirm the exact scenario.
In our design, under green light induction, the pSR43.6-pSR58.6-CheA and pSR43.6-pSR58.6-CheY systems would overexpress the CheA and CheY proteins, respectively. This would increase CheY-A production, enhance tumbling motility, and weaken straight-line motility of the bacteria. On semi-solid medium, this would manifest as reduced diffusive motility and a slower diffusion rate—specifically, forming smaller bacterial lawn areas within the same time period.
Figure 13 Results of semi-solid medium culture for the pSR43.6-pSR58.6-CheY system
Figure 14 Results of semi-solid medium culture for the pSR43.6-pSR58.6-CheA system
From the results in Figures 13 and 14, there was no significant difference in colony size between the green light-induced groups and the non-induced groups. This confirms that the variations in colony size observed in the pBBR1-MCS-5 system were likely caused by experimental errors. However, this does not sufficiently prove that overexpression of CheA or CheY has no effect on flagellar tumbling motility. Similarly, while the colony results from CheZ overexpression in semi-solid medium do not fully confirm enhanced straight-line motility, they provide preliminary macroscopic evidence of a potential effect. Further experiments are needed to clarify the exact impact of these protein overexpressions.
Therefore, we conducted the following microscopic observation assay to investigate bacterial motility at the microscopic level.
Verification 2: Microscopic Investigation via Microscopic Field Observation
To directly identify changes in the motility of engineered strains from a microscopic perspective, we designed a microscopic observation assay to visualize the movement trajectories of E. coli in a liquid environment, as shown in the figures below.
Figure 15 Microscopic movement trajectories of strains from the pDawn-CheZ system
Figure 15 presents the movement trajectories of four representative E. coli cells selected for observation. Microscopic observations revealed that the strains overexpressing CheZ exhibited a noticeable enhancement in straight-line motility. Compared to the macroscopic observations from the semi-solid medium assay, this microscopic evidence further confirms our expected outcomes.
Figure 16 Microscopic movement trajectories of strains from the pSR43.6-pSR58.6-CheY system
The E. coli cells observed in Figure 16 were strains overexpressing CheY. Observations of their motility revealed a significant decrease in movement ability. This is consistent with our previous hypothesis that overexpression of CheY enhances tumbling motility. We propose that if E. coli exhibits strengthened tumbling motility and weakened straight-line motility, it would appear as slow in-place wriggling rather than rapid movement under microscopic observation. This further confirms the success of our strain engineering. The unexpected diffusion results from the earlier semi-solid medium assay may be attributed to the difficulty of detecting changes in E. coli flagellar motility using macroscopic observation methods.
Section 4: Verification Assay for the Infectivity of Edwardsiella Piscicida
Figure 17 Infection assay of Edwardsiella piscicida labeled with red fluorescent protein
Figure 18 Infection assay of Edwardsiella piscicida labeled with green fluorescent protein
In this set of experiments, we constructed E. piscicida strains labeled with red fluorescent protein and green fluorescent protein, respectively, and subjected them to attenuation treatment before conducting infection assays. Through immersion infection assays on zebrafish larvae, we finally detected the infection efficiency using fluorescence microscopy, as shown in Figures 17 and 18. Observations revealed that the vaccine strains primarily accumulated in the gills, digestive tract, and skin surface of the zebrafish larvae. This is consistent with findings from previously reviewed literature, which indicate that vaccine strains typically enter the fish body through these three pathways and then reach immune organs such as the liver via bodily fluids to activate the immune response—thereby exerting their function as a vaccine.
Section 5: Conclusion and Outlook
Conclusion
Our goal was to design a light-inducible system for precise control of Edwardsiella piscicida motility. This would allow accurate immersion vaccination in fish. As a first step, we performed proof-of-concept studies in E. coli.
Using pBBR1-MCS-5, we showed that CheZ, CheA, and CheY can alter flagellar behavior. We then used pDawn and pSR43.6r&pSR58.6 systems to achieve light-inducible control. Under blue light, cells were expected to swim linearly. Under green light, cells were expected to tumble.
We successfully constructed 13 engineered strains. PCR and gel electrophoresis confirmed correct assembly. Western blotting verified protein expression. Semi-solid assays and microscopy demonstrated changes in motility consistent with our design. sfGFP served as a reporter to measure light-inducible expression levels. These data supported our prediction of motility regulation.
To test translational application, we performed zebrafish immersion infection with fluorescently labeled Edwardsiella piscicida. Bacteria localized to major entry points and immune organs. This confirmed the feasibility of our proposed vaccination concept.
Outlook
Time limits restricted our current work to concept validation. In the future, we will:
- Repeat experiments to ensure reproducibility.
- Combine blue- and green-light-inducible systems in a single strain. This will allow ratio-dependent control of linear versus tumbling motility.
- Transfer the system to Edwardsiella piscicida. This should enable precise, light-controlled infection during immersion vaccination. The strategy may improve efficiency, dosage control, and delivery accuracy in aquaculture vaccines.