Construction of fusion protein

We constructed our plasmid using homologous recombination. After lac operon expression in the vector, we designed homologous arms at both ends of the target gene and used homologous recombinase to introduce the target gene into the plasmid backbone, thereby constructing pHT01-tasA plasmid. The pHT01-tasA plasmid was then used as a plasmid scaffold to construct the TasA fusion protein plasmid.

We selected Bacillus subtilis with both endogenous tasA and sinR sequences knocked out as our engineered strain. Since the constitutive promoter plasmid carrying exogenous TasA protein exhibits toxicity to the engineered bacteria and maintains continuous expression of the foreign protein, which adversely affects growth, we designed an IPTG-inducible promoter (lac operon) preceding the exogenous tasA sequence.

Fig 2.1-1: pHT01-tasA plasmid and other TasA fusion protein plasmids.

Fig 2.1-2: Sequencing results of pHT01-tasA plasmid and other TasA fusion protein plasmids.

Sequencing results of pHT01-tasA and other TasA fusion protein particles showed that the sequencing results of key regions were completely consistent with standard gene sequences, with no mutations occurring. pHT01-tasA and other TasA fusion protein particles have been successfully constructed, and the bacterial strains were preserved for functional validation.

pHT01 plasmid functional verification

pHT01-TasA-mcherry function verification

To explore the function of the TasA fusion protein, we introduced the pHT01-TasA-mcherry plasmid into knockout bacteria, added 1mM IPTG, and induced expression at 37℃for 48 hours. The fluorescence expression results were observed under a fluorescence microscope.

Fig 2.1-3: Fluorescence microscope examination results.
Left picture: Observation under a fluorescence microscope in a bright field; Right picture: Observation under green excitation light of a fluorescence microscope.

According to the results of fluorescence microscopy examination, the TasA-MCherry fusion protein expressed by \(\textit{tasA}^-\textit{sinR}^-\)/tasA-mcherry after IPTG induction can emit red fluorescence, indicating that the TasA fusion protein we constructed can be expressed in Bacillus subtilis.

TasA fusion protein Congo red staining

To further verify the function of the TasA fusion protein we constructed, we performed cone red staining on the TasA protein on the surface of biofilms with wild-type Bacillus subtilis 168 strain(WT) introduced, Bacillus subtilis 168 strain with tasA and sinR genes (\(\textit{tasA}^-\textit{sinR}^-\)) knocked out, and knockout bacteria with pHT01-tasA-mfp5、pHT01-tasA-PS tag1、pHT01-tasA-PS tag2 (\(\textit{tasA}^-\textit{sinR}^-\)/tasA-mfp5,\(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag1,\(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag1) plasmid introduced. Observe the expression of the fusion protein.

Fig 2.1-4: TasA protein Congo red measurements. W = OD502/Quality conversion coefficient.
Control group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA strain; Positive control group: WT strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA-mfp5 strain, \(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag1 strain, \(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag2 strain; Three repetitions in each group.

According to the results of Congo red staining of TasA protein, the control group with the tasA gene knocked out basically did not express TasA protein, while the engineered bacteria introduced with pHT01-tasA-PS-tag1 and pHT01-tasA-PS-tag2 plasmids expressed a higher content of TasA protein. It indicates that the fusion protein plasmid we constructed has a strong ability to express fusion proteins. The amount of TasA protein expressed by Bacillus subtilis with the pHT01-tasA-mfp5 plasmid introduced in the figure is less than that of the wild type. This might be due to the excessive molecular weight of the TasA-Mfp5 fusion protein, which has a certain impact on the secretion and assembly of the protein.

Verification of the adhesion function of TasA protein

In order to investigate the function of the pHT01-tasA plasmid, we cultured three strains: wild-type Bacillus subtilis 168 (WT), the tasA-sinR mutant strain (\(\textit{tasA}^-\textit{sinR}^-\)), and the \(\textit{tasA}^-\textit{sinR}^-\)/tasA knockout strain with the pHT01-tasA plasmid. The cultures were incubated for 24 hours in polystyrene microspheres. Following ultrasonic washing to detach adherent bacteria, the bacterial suspension was diluted with PBS and spread on agar plates for colony-forming unit (CFU) counting.

CFU counts revealed that Bacillus subtilis strain 168 exhibited significantly reduced adhesion capacity after tasA gene knockout. However, the introduction of exogenous tasA gene partially offset this impairment. Strains with exogenous tasA expression still showed lower CFU counts compared to wild-type 168 strains, likely due to insufficient levels of induced TasA protein production.

Fig 2.1-5: Schematic diagram of dilution and coating of three types of bacteria.

Fig 2.1-6: CFU count bar chart.
Control group: \(\textit{tasA}^-\textit{sinR}^-\) strain; Positive control group: WT strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA strain; Three repetitions in each group.

Meanwhile, we performed crystal violet staining on the Bacillus subtilis adhering to the polystyrene spheres. By measuring the changes in the OD570 absorbance of the bacterial liquid eluted from the small ball filter material, the influence of different strains on adhesion was further analyzed.

In the two bacterial crystal violet staining experiments, it can be seen that the OD570 measured by Bacillus subtilis with the exogenous tasA gene introduced was higher than that of the control group. The results indicate that the adhesion ability of Bacillus subtilis with the exogenous tasA gene introduced has been improved to a certain extent, and the exogenous TasA protein can enhance the adhesion ability of Bacillus subtilis to a certain extent.

Fig 2.1-7: Comparison of crystal violet staining results of two types of bacteria.
Control group: \(\textit{tasA}^-\textit{sinR}^-\) strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA strain; Three repetitions in each group.

Adhesion force test

To test the adhesion ability of the TasA fusion protein, we introduced knockout bacteria of the plasmids pHT01-tasA-Mfp5, pHT01-tasA-PS tag1, and pHT01-tasA-PS tag2 (\(\textit{tasA}^-\textit{sinR}^-\)/tasA-Mfp5, \(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag1, \(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag1) and incubated them with polystyrene microsphere filter media for 24 hours respectively. The bacteria adhering to the polystyrene beads were ultrasonically eluted and then diluted with PBS for coating. CFU counts were conducted and compared with knockout bacteria introduced with the pHT01-tasA plasmid (\(\textit{tasA}^-\textit{sinR}^-\)/tasA)for comparative analysis.

It can be known from the CFU count results that the adhesion force of Bacillus subtilis introduced with TasA fusion protein granules has been significantly improved compared with the \(\textit{tasA}^-\textit{sinR}^-\)/tasA strain. The adhesion of Bacillus subtilis introduced with the TasA-Mfp5 fusion protein plasmid was stronger than that of Bacillus subtilis introduced with the TasA-PS tag1/TasA-PS tag2 fusion protein plasmid.

Fig 2.1-8: Schematic diagram of dilution and coating of granular bacteria containing different TasA fusion proteins.

Fig 2.1-9: Bar chart of CFU counts for strains containing different fusion protein granules.
Control group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA-mfp5 strain, \(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag1 strain, \(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag2 strain; Three repetitions in each group.

Meanwhile, we performed crystal violet staining on the Bacillus subtilis adhering to the polystyrene spheres. By measuring the changes in the \(\text{OD}_{570}\) absorbance of the bacterial liquid eluted from the small ball filter material, the differences in adhesion forces of different fusion proteins were further analyzed. The TasA-Mfp5 fusion protein has a stronger adhesion ability compared to the TasA-PS tag1/TasA-PS tag2 fusion protein. Considering that the Mfp5 adhesion protein has a wider range of adhesion activities, we chose the TasA-Mfp5 fusion protein for verification in the subsequent experiments.

Fig 2.1-10: Comparison of crystal violet staining results of granulosa containing different TasA fusion proteins.
Control group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA-mfp5 strain, \(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag1 strain, \(\textit{tasA}^-\textit{sinR}^-\)/tasA-PS tag2 strain; Three repetitions in each group.

During the CFU counting experiment, the number of adherents of TasA-PS tag1 was higher than that of TasA-PS tag2, but the amount of biofilm adhered by TasA-PS tag1 was greater than that of TasA-PS tag2. The difference in adhesion exhibited by the fusion protein It might be because the environment during the crystal violet staining and elution process has a significant impact on the structure of PS-tag1, resulting in a lower \(\text{OD}_{570}\) of the TasA-PS tag1 fusion protein in Figure 2.1-11.

Detection of adhesion force of fusion proteins in sewage environment

Due to the high concentration of heavy metal ions and low environmental pH in the heavy metal wastewater treatment environment, in order to ensure that Bacillus subtilis introduced with TasA-Mfp5 fusion protein still exerts adhesion in the wastewater environment, we simulated environmental conditions with different \(\text{Cd}^{2+}\) concentrations and lower pH values. To explore the influence of different environments on the adhesion of the TasA-Mfp5 fusion protein.

We designed SYG media containing different \(\text{Cd}^{2+}\) concentrations (0 mg/L, 0.25 mg/L, 0.5 mg/L, 1 mg/L, 2 mg/L), and incubated the knockout bacteria introduced with pHT01-tasA-mfp5 with polystyrene microsphere filter media for 48 hours. The bacteria adhering to the polystyrene beads were ultrasonically eluted and then diluted with PBS and coated for CFU counting.

Fig 2.1-11: Schematic diagram of dilution and coating of TasA-Mfp5 fusion protein bacteria at different \(\text{Cd}^{2+}\) concentrations.

Fig 2.1-12: Bar charts of CFU counts of TasA-Mfp5 fusion protein bacteria at different \(\text{Cd}^{2+}\) concentrations.

It can be known from CFU counts that within a certain range of \(\text{Cd}^{2+}\) concentrations (0-0.5mg/L), with the increase of \(\text{Cd}^{2+}\) concentration, the adhesion ability of the TasA-Mfp5 fusion protein decreases to a certain extent, but there were no significant differences.

We simultaneously designed SYG media with different pH values (pH=2, 3, 4, 5, 6, 7). The knockout bacteria introduced with pHT01-tasA-mfp5 were incubated and cultured with polystyrene microsphere filter media for 48 hours. The bacteria adhering to the polystyrene microspheres were ultrasonically eluted and then diluted with PBS and spread for CFU counting.

Fig 2.1-13: Schematic diagram of dilution and coating of TasA-Mfp5 fusion protein bacteria under different environmental pH conditions.

Fig 2.1-14: Bar charts of CFU counts of TasA-Mfp5 fusion protein bacteria under different environmental pH conditions.

It can be known from the CFU count that when the pH value of the living environment is too low (pH=2), the growth of TasA-Mfp5 fusion protein bacteria is significantly inhibited. With the increase of pH (pH=3), the inhibitory effect of environmental pH on the growth of TasA-Mfp5 fusion protein bacteria was relieved to a certain extent; In a weakly acidic environment (pH=4-6), there was no significant difference in the CFU count of TasA-Mfp5 fusion protein bacteria, and the adhesion ability was less affected by the environmental PH. When pH= 7, the CFU count of TasA-Mfp5 fusion protein bacteria decreased significantly. This might be because the surface of the polystyrene microspheres incubated with the bacterial liquid was damaged during the experiment, and the adhesion process with the fusion protein was affected.

Background and Introduction

SpyTag is a short peptide that forms an isopeptide bond upon encountering its protein partner SpyCatcher. This covalent peptide interaction is a simple and powerful tool for bioconjugation and extending what protein architectures are accessible. Originated from fibronectin binding protein FbaB, SpyTag-SpyCatcher exhibit spontaneous reconstruction ability under a wide range of pH values, redox conditions and temperatures, which can perfectly fit in harsh application conditions. In the practical application of the TasAnchor system, there may be issues with the TasA fusion protein being relatively large, making it difficult to express and secrete. By inserting SpyTag-SpyCatcher into our adhesion system, we can further improve the modularity of TasAnchor, make it easier to adapt to various filter materials.

Plasmid Construction

SpyTag-SpyCatcher Interaction Experiment

Adhesion Function Examination

Conclusion

Construction and Verification of the pHT01-epcadR-pcadR-mcherry Plasmid

Using mcherry as the reporter gene, the plasmid pHT01-epcadR-pcadR-mcherry was constructed to evaluate the response of the epcadR-pcadR module to environmental cadmium ion concentrations. Sequencing results confirmed the successful assembly of all functional elements.

Fig 3.1-1: Plasmid map of pHT01-epcadR-pcadR-mcherry.

Fig 3.1-2: Sequencing result of pHT01-epcadR-pcadR-mcherry.

The pHT01-epcadR-pcadR-mcherry plasmid was introduced into the ΔtasAΔsinR Bacillus subtilis 168 strain using a competent cell preparation kit. Colony PCR confirmed the successful introduction of the recombinant plasmid.

Fig 3.1-3: Colony PCR was performed to verify the successful introduction of the pHT01-epcadR-pcadR-mcherry recombinant plasmid.

Growth Curves of Plasmid-Free Knockout Strains and Engineered Strains under Different Cadmium Ion Concentrations

Fig 3.1-4: Growth curves of the two strains under different cadmium ion concentrations.

The results indicate that, at cadmium ion concentrations measured in mg/L, both strains exhibited rapid growth, suggesting minimal inhibitory effects at these levels. The strongest inhibition was observed at 1.5 mg/L, while 0.5 mg/L and 0.25 mg/L showed relatively weak inhibition with little difference between them. Overall, cadmium ions at the 0-1.5 mg/L scale exert only minor effects on the growth of the engineered strain, which is capable of robust growth under the experimental cadmium concentrations. Furthermore, comparison of the two strains indicates that the introduction of the exogenous plasmid has negligible impact on the cadmium tolerance of the engineered strain.

Fluorescence Response Curves of Engineered Strains to Different Cadmium Ion Concentrations over Time

Fig 3.1-5: Time-course of fluorescence intensity in engineered strains under different cadmium ion concentrations.

Under treatments with 0, 1, and 2 mmol/L cadmium ions, fluorescence intensity in the pHT01-epcadR-pcadR-mcherry strain showed a significant positive correlation with both cadmium concentration and incubation time. The strongest fluorescence was observed at 2 mmol/L, reaching a peak at 25 h, while the 0 mmol/L control group maintained a consistently low fluorescence level (less than 30 FU), demonstrating that cadmium ions effectively induce fluorescence expression.

Construction and Verification of the pHT01-tasA-smtA Plasmid

We first used the pHT01-tasA plasmid as a template for reverse PCR to obtain the vector backbone. Subsequently, the smtA fragment was joined to this backbone via Gibson assembly, resulting in the successful construction of the pHT01-tasA-smtA recombinant plasmid. Sequencing results confirmed the correct assembly of all functional elements.

Fig 3.2-1: Plasmid map of pHT01-tasA-smtA.

Fig 3.2-2: Sequencing result of pHT01-tasA-smtA.

The pHT01-tasA-smtA plasmid was introduced into the ΔtasAΔsinR Bacillus subtilis 168 strain using a competent cell preparation kit. Colony PCR confirmed the successful introduction of the recombinant plasmid.

Fig 3.2-3: Colony PCR was performed to verify the successful introduction of the pHT01-tasA-smtA recombinant plasmid.

Effects of Cadmium Ions on Biofilm Formation in Engineered Strains

Fig 3.2-4: Results of Crystal Violet Staining of Biofilms Formed by Engineered Strains under Different Cadmium Ion Concentrations.

TasA is a key structural protein involved in biofilm formation and a major component of the extracellular matrix; therefore, the metal ion adsorption capacity of the TasA-SmtA fusion protein is closely associated with the biofilm formation status of the engineered strain. To ensure that this adsorption module functions under normal biofilm conditions, we examined the effects of different cadmium ion concentrations on biofilm formation in the engineered strain. Strains carrying pHT01-tasA-smtA were cultured under varying cadmium ion concentrations, and after 36 h of incubation, the formed biofilms were stained with crystal violet. Biofilm production was quantified by measuring OD₅₇₀. The results showed that within the cadmium concentration range of 0-9 mg/L, biofilm formation of the pHT01-tasA-smtA strain was not significantly affected. These findings indicate that the TasA-SmtA module can stably form biofilm across a wide range of environmental cadmium concentrations.

Adsorption-desorption Curves of Cadmium Ions by Engineered Strains

SmtA protein forms reversible complexes with Cd²⁺ ions through metal-coordinating sites such as thiol and carboxyl groups. Under high pH conditions, these sites are deprotonated, facilitating Cd²⁺ adsorption, whereas under low pH conditions, protonation promotes Cd²⁺ desorption, enabling controlled release of the metal ions. Based on this mechanism, adsorption-desorption curves of the engineered strain were measured in 9 mg/L and 5 mg/L Cd²⁺ solutions, demonstrating its potential for repeated use.

Fig 3.2-5: Adsorption-desorption Curves of Cadmium Ions by the pHT01-tasA-smtA Engineered Strain.

The engineered strain underwent sequential adsorption-desorption cycles, repeated three times over a total of two rounds. As shown in the figure, for the 9 mg/L Cd²⁺ solution, the pHT01-tasA-smtA strain maintained high and stable adsorption capacity throughout the cycles. The initial adsorption nearly achieved 100% removal of cadmium ions. Although desorption did not remove all bound Cd²⁺, the engineered strain still exhibited substantial adsorption efficiency.

For the 5 mg/L Cd²⁺ solution, the initial adsorption efficiency was relatively low; however, the strain demonstrated 100% desorption efficiency during the cycles, indicating good regenerability. Moreover, adsorption efficiency increased noticeably after the first desorption, which may be attributed to activation of SmtA proteins displayed on the cell surface under low pH conditions.

The cadmium ion adsorption efficiency of the pHT01-tasA-smtA engineered strain was evaluated using a 9 mg/L Cd²⁺ solution. The results showed that during the initial adsorption, the strain achieved a 100% removal rate, effectively adsorbing all Cd²⁺ in the solution. After two rounds of desorption under low pH conditions, the adsorption efficiency decreased but remained at approximately 32%. These findings indicate that the engineered strain not only efficiently removes cadmium ions from the environment but also retains substantial adsorption activity after multiple desorption cycles, demonstrating strong potential for regeneration and reuse.

Construction and Verification of the Sensing-adhesion Composite Plasmid

To enable the engineered strain to adhere to biofilter materials and perform cadmium ion sensing, a dual-module “sensing-adhesion” expression plasmid was constructed. Preliminary experiments indicated that among various candidate adhesion proteins, Mfp5 exhibited the most favorable surface-binding performance. Compared with the SpyTag-SpyCatcher system, the TasA-Mfp5 system offered a simpler construction process without requiring additional protein purification steps. Based on these advantages, the TasA-Mfp5 adhesion module was integrated with the epcadR-pcadR-mcherry sensing module to evaluate the cadmium-responsive capability of the engineered strain in an attached state.

To enhance overall expression efficiency, the strong constitutive promoter p43 was used to replace the original promoter sequence. Subsequently, the recombinant plasmid pHT01-p43-epcadR-mcherry-p43-tasA-mfp5 was successfully obtained via homologous recombination. This plasmid endows the host strain with stable adhesion to polystyrene microspheres and enables a fluorescent response in the presence of exogenous cadmium ions.

Sequencing verification confirmed the correct construction of the target plasmid, providing an experimental foundation for subsequent quantitative detection and characterization of cadmium ion responsiveness.

Fig 3.3-1: Plasmid map of pHT01-p43-epcadR-mcherry-p43-tasA-mfp5.

Fig 3.3-2: Sequencing result of pHT01-p43-epcadR-mcherry-p43-tasA-mfp5.

The pHT01-p43-epcadR-mcherry-p43-tasA-mfp5 plasmid was introduced into the ΔtasAΔsinR Bacillus subtilis 168 strain using a competent cell preparation kit. Colony PCR confirmed the successful introduction of the recombinant plasmid.

Fig 3.3-3: Colony PCR was performed to verify the successful introduction of the pHT01-p43-epcadR-mcherry-p43-tasA-mfp5 plasmid into the ΔtasAΔsinR Bacillus subtilis 168 strain.

Verification of Fluorescence Expression under the Adherent State

In the experiment, we incubated the polystyrene globules with the SYG film-forming medium of the engineered bacteria for 30 h, then used the incubated globules for cadmium ion adsorption experiments, and then incubated with 2mmol/L cadmium ion solution for 16h, and then observe fluorescence under a microscope.

Fig 3.3-4: Fluorescence Microscopy Observation of ΔtasAΔsinR Bacillus subtilis 168 Strain Carrying pHT01-p43-epcadR-mcherry-p43-tasA-mfp5 and the ΔtasAΔsinR Bacillus subtilis 168 Strain.

Fig 3.3-5: Observation of Polystyrene Microspheres under Yellow Light (leftmost: ΔtasAΔsinR Bacillus subtilis 168 carrying pHT01-p43-epcadR-mcherry-p43-tasA-mfp5; second from left: ΔtasAΔsinR Bacillus subtilis 168).

Polystyrene microspheres with adhered pHT01-p43-epcadR-mcherry-p43-tasA-mfp5 cells exhibited pronounced red fluorescence under a fluorescence microscope after induction with 2 mmol/L cadmium chloride, the concentration producing maximal fluorescence. The fluorescence was concentrated on the bacterial cells. From a macroscopic perspective, under yellow light, microspheres carrying the pHT01-p43-epcadR-mcherry-p43-tasA-mfp5 strain appeared redder compared to those with the ΔtasAΔsinR strain.

These results demonstrate that the pHT01-p43-epcadR-mcherry-p43-tasA-mfp5 strain can simultaneously adhere to polystyrene microspheres and respond to cadmium ions by expressing fluorescence, confirming the successful construction of the engineered strain with a dual “sensing-adhesion” functional module.