E. coli DH5α was purchased from Takara (Japan). First, the thiosulfate operon sequence was synthesized, coupled with mRFP, and cloned into the pSB1A3 plasmid (Genewiz, USA) to obtain pSB-PphsA-mRFP. After sequence verification (Qingke, China), the plasmid was extracted using a plasmid extraction kit (Tiangen, China). The recombinant plasmid was then transformed into E. coli DH5α. The engineered strain was cultured in LB medium containing ampicillin (Amp) at a concentration of 100 μg/mL at 37℃.
The engineered bacteria were inoculated into fresh LB medium containing different concentrations of sodium thiosulfate pentahydrate (Sorlabio, S5280) and cultured for 8 hours. A microplate reader (Multiskan GO, Thermo Fisher Scientific, USA) was used to measure the OD600 value (absorbance at 600 nm), and the OD600 was then adjusted to around 1 using LB medium. The microplate reader was subsequently used to measure fluorescence values (excitation wavelength 584 nm, emission wavelength 607 nm) and OD600 values. The normalized fluorescence ratio (Fluorescence/OD600) was then calculated.
The cultures were inoculated into LB medium containing 1 mM of sodium sulfate (MACKLIN, 7757-82-6), sodium sulfite anhydrous (MACKLIN, 7757-83-7), sodium tetrathionate dihydrate (MACKLIN, 13721-29-4), and thiosulfate, then incubated for 8 hours. Afterward, the OD600 was measured using a microplate reader and adjusted to approximately 1. Fluorescence values (excitation wavelength 584 nm, emission wavelength 607 nm) and OD600 were then recorded. The normalized fluorescence ratio (Fluorescence/OD600) was calculated for further analysis.
The thiosulfate operon sequence was synthesized, linked to LacZα, and cloned into the pSB1A3 plasmid (Genewiz, USA) to construct pSB-PphsA-LacZα. After verifying the sequence (Qingke, China), the recombinant plasmid was transformed into E. coli DH5α. The engineered strain was then cultured at 37℃ in LB medium containing 100 μg/mL of ampicillin (Amp).
Overnight cultures were diluted 1:100 into fresh LB medium with varying concentrations of thiosulfate and incubated at 37℃ until reaching the logarithmic growth phase (OD600 = 0.6). A 1 mL sample of the bacterial culture was collected into a centrifuge tube, and 1 mL of lysis buffer was added. The cells were disrupted by ultrasonic treatment on ice (200 W power, 3 s ultrasonication, 10 s intervals, repeated 30 times). The lysate was centrifuged at 15,000 g for 20 minutes at 4℃, and the supernatant was collected and kept on ice for further analysis. β-galactosidase activity was measured using a β-galactosidase reporter gene detection kit (AKSU042M, boxbio). After a 30-minute reaction, the OD400 of the samples was recorded, and a standard curve was generated using the standard solution. The enzyme activity unit was defined as OD600 = 1 bacteria producing 1 nM p-Nitrophenyl-β-D-galactopyranoside (ONPG) per hour.
To test the ability of engineered strains to degrade X-gal via β-galactosidase activity, overnight cultures were diluted 1:100 into fresh LB medium containing different concentrations of thiosulfate and incubated at 37°C until the bacteria reached the logarithmic growth phase (OD600 = 0.6). X-gal was dissolved in dimethyl sulfoxide (DMSO). A 1 mL sample of the bacterial culture was collected into a centrifuge tube, and an appropriate amount of X-gal was added. The culture was incubated at 37°C, and color changes were observed to determine β-galactosidase activity, which indicates the degradation of X-gal.
E. coli Nissle 1917 (EcN) was purchased from Biobw (China). A recombinant plasmid, pSB-PEA (Genewiz, USA), was then synthesized. This plasmid includes the thiosulfate-responsive ThsS/R system (with promoters Pj23104/Pj23100), Hly-EGF (under the PphsA promoter), and the α-hemolysin secretion system (hlyB and hlyD) (driven by promoter Pj23104). In parallel, a control plasmid, pSB-PE, lacking the α-hemolysin secretion system, was also synthesized. The recombinant plasmids were subsequently transformed into E. coli DH5α and EcN, respectively. The engineered strains were cultured at 37℃ in LB medium containing 100 μg/mL of ampicillin (Amp).
EcN expressing hEGF was inoculated into 50 mL of LB medium containing different concentrations of sodium thiosulfate (0 mM, 1 mM) and cultured overnight. The following day, 5 mL of the bacterial culture was collected, adjusted to OD600 = 1, and centrifuged at 10,000 x g for 1 minute. The supernatant was transferred to a new centrifuge tube to serve as the extracellular component sample. The bacterial pellet was resuspended in 1 mL of PBS and lysed by ultrasonic disruption (75 W, 1 s ultrasonic pulse, 3 s intervals, for a total of 20 minutes). The lysed cell suspension was transferred to a new centrifuge tube as the intracellular component sample. Finally, the hEGF content was measured using an ELISA kit (mlbio, China).
EcN expressing hEGF was inoculated into 50 mL of LB medium containing 1 mM sodium thiosulfate and cultured overnight. The following day, 5 mL of the bacterial culture was collected and adjusted to OD600 = 1. The culture supernatant and bacterial content samples were obtained through ultrasonic disruption and centrifugation. EcN containing only an empty vector served as a control. Human embryonic kidney 293T cells were seeded into 24-well plates at a density of 5×10^5 cells per well in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were cultured at 37℃ in a humidified atmosphere with 5% CO2 until they reached 70% confluence. Then, 100 μL of the bacterial content and filtered supernatant from the engineered bacteria were added to the DMEM medium. After 8 hours of incubation, cell viability was assessed using the CCK8 assay. Specifically, 100 µL of CCK8 solution was added to each well and incubated at 37℃ for 3 hours. Absorbance at 450 nm was then measured using a microplate reader.
The sequence of the rhamnose-inducible promoter PRha and the ribosome binding site (RBS) B0034 was obtained through gene synthesis and cloned into the pSB1A3 vector at the XbaI and SpeI restriction sites, upstream of the fluorescent protein gene mRFP. The recombinant plasmid was then transformed into E. coli BL21 using the calcium chloride heat-shock method.
A 1:100 dilution of the rhamnose biosensor strain was inoculated into LB medium and cultured overnight at 37℃. The next day, the overnight culture was diluted 1:50 into fresh M9 medium containing 50 μg/mL ampicillin and supplemented with 0.4% glucose. Simultaneously, a rhamnose solution at concentrations ranging from 0.1% to 1% was added to the medium. The culture was induced at 37℃ for 5 hours, and 1 mL of the culture was sampled. OD600 and fluorescence values (excitation wavelength 584 nm, emission wavelength 607 nm) were measured using a microplate reader. The normalized fluorescence ratio (Fluorescence/OD600) was calculated.
The mazF and mazE genes were synthesized and cloned into the pSB1A3 recombinant vector containing the rhamnose promoter. The antitoxin protein gene mazE was placed downstream of the pRha-B0034 promoter and regulated by rhamnose, while the toxin protein gene mazF was placed downstream of the pBAD promoter and regulated by arabinose. After sequencing verification of the recombinant plasmid, it was transformed into E. coli BL21.
A 1:100 dilution of the engineered BL21 strain containing the rhamnose-responsive suicide system was inoculated into LB medium and cultured overnight at 37℃. The next day, the overnight culture was diluted 1:50 into fresh M9 medium containing 50 μg/mL ampicillin, 1% rhamnose, and 0.2% arabinose, supplemented with 0.4% glucose. The OD600 values were monitored over time using a microplate reader.
Data were analyzed and visualized using GraphPad Prism software. Results are presented as mean ± standard deviation (SD). One-way ANOVA followed by Tukey’s post hoc test was performed to analyze differences between groups. A p-value of less than 0.05 was considered statistically significant.
