Based on the plasmid maps, the NETMAP NIR light–inducible regulatory module was synthesized (Generalbiol, China). NETMAP comprises two plasmids. Plasmid A is built on the pSB4C5 backbone, carries chloramphenicol resistance, constitutively expresses YhjH and MrkH from the tac promoter, and includes the PmrkA-mRFP cassette. Plasmid B is built on the pSB1A3 backbone, carries ampicillin resistance, and constitutively expresses PadC4 and BphO from the lac promoter. The synthesized sequences were inserted upstream of the mRFP coding region in the pSB1A3 vector using one-step seamless cloning (Seamless Cloning Kit, D7010, Beyotime). The recombinant plasmids were then introduced into E. coli DH5α and BL21 by heat shock (42 °C, 1 min). Positive clones were selected on LB agar plates (1.5% agar) containing ampicillin (100 μg/mL) and chloramphenicol (30 μg/mL) and verified by Sanger sequencing (Tsingke, Beijing), yielding the engineered strains DH5α-NETMAP-mRFP and BL21-NETMAP-mRFP. Strains were stored at −20 °C with 25% (v/v) glycerol as cryoprotectant. Unless otherwise noted, cultures were grown at 37 °C with shaking at 150 rpm, and inoculation/scale-up was performed in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL ampicillin.
To evaluate NIR light–induced gene expression, engineered bacterial strains carrying the NETMAP–mRFP construct were inoculated at a 1:100 ratio into 5 mL of LB medium supplemented with 100 μg/mL ampicillin (Amp) and 30 μg/mL chloramphenicol (Cm). The 15 mL centrifuge tubes were wrapped in aluminum foil to prevent unwanted light exposure, and cultures were incubated at 37 °C with shaking at 180 rpm for 12 hours. After the overnight growth, the bacterial cells were reinoculated at a 1% (v/v) ratio into a 48-well plate containing 500 μL of LB medium supplemented with Amp and Cm, and incubated continuously at 37 °C and 180 rpm until the optical density reached OD₆₀₀ = 0.5. Subsequently, the cultures were irradiated with 660 nm near-infrared (NIR) light using a light-emitting diode (LED) source for 3 hours to induce gene expression.
To evaluate the effect of illumination duration on the NETMAP system, samples were exposed to 660 nm NIR light for varying time periods (0–8 hours). A total of 100 μL of bacterial culture was transferred to a 96-well plate, and both OD₆₀₀ and mRFP fluorescence intensity were measured using a microplate reader (FlexStation 3, Molecular Devices, USA) with an excitation wavelength of 584 nm and an emission wavelength of 607 nm. The normalized fluorescence intensity of each sample was calculated as the ratio of fluorescence intensity to OD₆₀₀ (fluorescence/OD₆₀₀).
Engineered bacteria carrying NETMAP–mRFP were cultured until the OD₆₀₀ reached approximately 0.5. One group was maintained under dark conditions, while the other was continuously irradiated with 660 nm NIR light for 3 hours. Following induction, 100 μL of each culture was transferred to a 96-well plate or flow cytometry tube for dilution and sample preparation according to standard flow cytometry protocols. Single-cell fluorescence was detected using a flow cytometer (FlexStation 3, Molecular Devices, USA) to assess fluorescence differences between the dark and light-irradiated groups. Data were collected and analyzed via the PC5.5-A channel (the mRFP fluorescence channel, detecting emission around 607 nm), and the fluorescence intensity distribution of the bacterial population was presented in the form of histograms to visualize population-level expression profiles.
The coding genes for Coa, YopE1-15 PD-L1 nanobody, and INP-HlpA were synthesized and codon-optimized for E. coli. Restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to comply with RFC#10 standards and pET28a (m) vector cloning requirements. Each gene (Coa, YopE1-15 PD-L1 nb, and INP-HlpA) was inserted into the pET28a (m) vector via NdeI and XhoI restriction sites. The recombinant plasmids were then transformed into competent E. coli BL21 cells using the heat shock method (42°C, 1 min). Positive transformants were selected on LB agar plates (supplemented with 1.5% agar) containing 100 μg/mL kanamycin (Kana) and confirmed by Sanger sequencing (Tsingke, Beijing). The recombinant strains BL21–Coa, BL21–YopE1-15 PD-L1 nb, and BL21–INP-HlpA were successfully obtained. Strains were stored at –20°C in 25% (v/v) glycerol as a cryoprotectant. For cultivation, engineered strains were grown in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin at 37°C and 150 rpm.
For protein induction, frozen bacterial cultures stored at –80°C were inoculated at a 1:100 ratio into 5 mL of LB broth (supplemented with 100 μg/mL kanamycin) in a 15 mL centrifuge tube and cultured overnight at 37°C with shaking at 150 rpm. Then, 1% (v/v) of the culture was re-inoculated into 30 mL of fresh LB broth (A507002, Sangon Biotech) and cultured at 37°C and 180 rpm for 1–2 hours. When the OD₆₀₀ reached 0.2, 0.5 mM IPTG was added to induce protein expression. The culture temperature was adjusted to 16°C, and incubation continued for 20 hours to enhance soluble protein production.
After fermentation, 5 mL of culture was collected by centrifugation (10,000 × g, 5 min). The bacterial pellet was resuspended in PBS, and ultrasonic disruption was performed on ice using an ultrasonic disruptor (Shanghai Jinxin, China) under the following conditions: 1 s on / 3 s off cycle, 70 W power, total 20 min. The lysate was centrifuged at 10,000 × g, 4°C for 30 min (Biosafer1000, Saifei), and the supernatant was collected as the intracellular protein sample. Protein concentration was determined using the Bradford Protein Assay Kit (P0006, Beyotime).
Protein samples were separated using a 12.5% SDS-PAGE gel prepared with the One-step PAGE Gel Rapid Preparation Kit (PG113, Yeasen). The separated proteins were transferred onto a 0.22 μm PVDF membrane (WJ001S, Yeasen) via wet transfer. The membrane was blocked at room temperature for 20 minutes with a protein-free rapid blocking buffer (ED0024, Sikejie), followed by incubation with a 1:1000-diluted mouse anti-His-tag antibody (AH367, Beyotime) as the primary antibody at 4°C overnight. After washing, the membrane was incubated with an HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (A0216, Beyotime) at room temperature for 1 hour. Finally, chemiluminescence detection was performed using a protein blot imaging system (ChemiDoc MP, Bio-Rad) to visualize the target protein bands.
The YopE1-15 PD-L1 nanobody (PD-L1 nb) gene (Generalbiol, China) was synthesized and codon-optimized for E. coli, with the EcoRI, XbaI, SpeI, and PstI restriction enzyme sites removed to comply with RFC#10 assembly standards. The mRFP reporter gene in the previously constructed NIR light–inducible biosensor (NETMAP) was replaced with the YopE1-15 PD-L1 nb sequence using the one-step seamless cloning method (Seamless Cloning Kit, D7010, Beyotime). The resulting recombinant plasmid was transformed into E. coli DH5α using the heat shock method (42°C, 1 min). Positive clones were screened on LB agar plates (supplemented with 1.5% agar) containing 100 μg/mL ampicillin (Amp) and verified by DNA sequencing (Tsingke, Beijing). The successfully engineered recombinant strain, DH5α–NETMAP–PD-L1 nb, was obtained and stored at –20°C in 25% (v/v) glycerol as a cryoprotectant. The engineered strain was cultured at 37°C with shaking at 150 rpm, and for inoculation and scale-up, LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL Amp was used as the growth medium.
The DH5α–NETMAP–PD-L1 nb strain was inoculated at a 1:100 ratio into 5 mL of LB medium containing 100 μg/mL ampicillin (Amp). The 15 mL centrifuge tubes were wrapped with aluminum foil to prevent light exposure. The culture was grown at 37°C and 180 rpm for 12 hours. Subsequently, the bacterial cells were inoculated at a 1% rate into 48-well plates containing 500 μL of Amp⁺ LB medium and further cultured at 37°C and 180 rpm until the OD₆₀₀ reached 0.5. At this point, samples were irradiated with 660 nm near-infrared light (NIR) using a light-emitting diode (LED) setup for 7 hours. After induction, the supernatant of the NETMAP-engineered bacterial culture was collected and centrifuged at 12,000 × g for 10 minutes at 4°C. The supernatant was then filtered through a 0.22 μm filter membrane, followed by concentration using a BeyoGold™ ultrafiltration tube (15 mL, 5 kDa MWCO, PES; FUF505, Beyotime) by centrifugation at 4,000 × g for 40 minutes at 4°C. The protein concentration of the concentrated sample was determined using the Bradford Assay Kit (P0006, Beyotime). For Western blot analysis, the concentrated protein samples were separated using a 12.5% SDS-PAGE gel prepared with the One-step PAGE Gel Rapid Preparation Kit (PG113, Yeasen). The proteins were then transferred onto a 0.22 μm PVDF membrane (WJ001S, Yeasen) via wet transfer. The membrane was blocked with a protein-free rapid blocking buffer (ED0024, Sikejie) at room temperature for 20 minutes, followed by incubation with a 1:1000-diluted mouse anti-His-tag antibody (AH367, Beyotime) as the primary antibody at 4°C overnight. After washing, the membrane was incubated with an HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (A0216, Beyotime) at room temperature for 1 hour.
For the preparation of recombinant strains, induced expression, and crude protein extraction: After codon optimization of the Coagulase-encoding gene and removal of restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI), the gene was cloned into the pET28a (m) vector via the NdeI and XhoI restriction enzyme sites. The recombinant plasmid was transformed into competent E. coli BL21 strain by the heat shock method (42°C, 1 min). Transformants were screened on LB solid plates containing 100 μg/mL kanamycin (Kana) to obtain the recombinant engineered strain BL21-Coa. After overnight culture of the BL21-Coa strain in LB broth containing Kana, it was inoculated at a ratio of 1:100 into 10 mL of fresh medium and cultured at 37°C with shaking at 180 rpm until the OD600 reached 0.5-0.8.
Non-anticoagulated blood samples were used. Subsequently, 50 μL of purified Coa protein solutions with different concentrations were mixed with 50 μL of blood samples in EP tubes or 96-well plates, with a total reaction volume of 100 μL. The concentration gradient of the Coa protein solution included 100%, 80%, 60%, 40%, and 20%, and a mixture of 0% Coa solution (i.e., buffer) and 50 μL of blood sample was used as the negative control. Meanwhile, 100 μL of water and 100 μL of 100% Coa solution were set as blank controls without blood. All mixtures were incubated in a 37°C incubator, and photos were taken every 5 minutes to record the coagulation status for 30 minutes. A final photo was taken at 60 minutes when complete coagulation was achieved. The main observation and analysis indicators included thrombosis formation time, thrombus morphology, and coagulation intensity.
A truncated ice nucleation protein (INP) was fused to the N-terminus of HlpA to display the HlpA protein on the surface of engineered probiotics. Additionally, mRFP was fused downstream of HlpA to enable visual observation of HlpA-mediated adhesion to colorectal cancer cells. The INP–HlpA–mRFP sequence (Generalbiol, China) was synthesized and codon-optimized for E. coli, with the EcoRI, XbaI, SpeI, and PstI restriction sites removed to comply with RFC#10 assembly standards. The J23100-B0034 cis-regulatory elements (constitutive promoter and RBS) were introduced upstream of the INP–HlpA sequence to drive continuous expression. The fusion gene was cloned into the pSB1A3 vector via the XbaI and SpeI restriction enzyme sites. The recombinant plasmid was transformed into E. coli BL21 using the heat shock method (42°C, 1 min). Positive clones were selected on LB agar plates (supplemented with 1.5% agar) containing 100 μg/mL ampicillin (Amp) and verified by Sanger sequencing (Tsingke, Beijing). The resulting recombinant strain, BL21–INP–HlpA, was obtained and stored at –20°C in 25% (v/v) glycerol as a cryoprotectant. The engineered strain was cultured at 37°C with shaking at 150 rpm, and inoculation and scale-up were performed in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL Amp.
To assess the adhesion ability of the engineered strain, CT26 colorectal cancer cells were seeded at a density of 2 × 10⁵ cells per well in a 6-well culture plate and incubated for 48 hours until reaching 80% confluence. The culture medium was replaced with fresh DMEM supplemented with 50 mg/L Amp. Subsequently, 1 × 10⁷ CFU of engineered probiotics were added and co-cultured with CT26 cells for 2 hours. After co-incubation, 100 μL of the supernatant was collected, serially diluted, and spread onto Amp-containing LB plates to determine the number of non-adherent (free) bacteria by CFU counting, allowing calculation of adhesion efficiency. The CT26 cells were washed twice with sterile PBS to remove unbound bacteria, and fluorescence images of adherent bacteria expressing mRFP were captured using an inverted fluorescence microscope to visualize bacterial adhesion on the cell surface.
As previously described, the E. coli toxin protein MazF was synthesized and codon-optimized to comply with RFC#10 standards. The MazF gene was placed downstream of the lactose-inducible promoter Plac, generating the construct Plac–mazF. Additionally, a LacI expression cassette (PlacI–LacI) was inserted upstream of Plac–mazF and separated by the transcriptional terminator B0015, resulting in the complete regulatory sequence PlacI–LacI–B0035–Plac–mazF. The assembled construct was cloned into the pSB1A3 vector via the XbaI and SpeI restriction sites, and the recombinant plasmid was transformed into E. coli DH5α by the heat shock method (42°C, 1 min).
Similarly, the E. coli toxin protein MazF was synthesized and codon-optimized according to RFC#10 standards. The MazF gene was placed under the control of the arabinose-inducible promoter PBAD, forming the construct PBAD–mazF. This sequence was cloned into the pSB1A3 vector via the XbaI and SpeI restriction sites and transformed into E. coli DH5α using the heat shock method (42°C, 1 min). Positive transformants were screened on LB agar plates (supplemented with 1.5% agar) containing 100 μg/mL ampicillin (Amp) and verified by DNA sequencing (Tsingke, Beijing). The successfully constructed recombinant strain, DH5α–PBAD–MazF, was stored at –20°C in 25% (v/v) glycerol as a cryoprotectant. The strain was cultured at 37°C with shaking at 150 rpm, and inoculation and expansion were performed in LB broth (G3102, Servicebio, China) containing 100 μg/mL Amp.
The frozen DH5α–PBAD–MazF strain stored at –20°C was revived by inoculating into 5 mL of LB medium containing Amp and cultured overnight at 37°C and 150 rpm. The overnight culture was then inoculated at a 1:100 ratio into 20 mL of fresh LB medium with Amp, and the OD₆₀₀ was adjusted to 0.1. One group was supplemented with 2% L-arabinose (A83229, Innochem) to induce MazF expression, while the control group was grown without arabinose. The growth curves of both groups were continuously monitored using a FlexStation 3 microplate reader (Molecular Devices, USA) to evaluate the effect of MazF induction on bacterial viability.
