Cytochrome P450 enzyme reductase (CrCPR) from Catharanthus roseus (Madagascar periwinkle), 2-hydroxyisoflavone synthase (2-HIS), also known as isoflavone synthase (LjIFS) from Lotus japonicus (bird's-foot trefoil), and 2-hydroxyisoflavanone dehydratase (GmHID) from Glycine max (soybean) were synthesized. Codon optimization for Escherichia coli was performed, and EcoRI, XbaI, SpeI, and PstI restriction sites were eliminated to comply with RFC#10 standards (Genewiz, USA). Using pSB1A3 as the vector and J23100-B0034 as the promoter-RBS sequence, a polycistronic sequence was constructed. A dual-promoter design was employed to enhance mRNA expression efficiency. The recombinant plasmid was transformed into E. coli BL21, and positive clones were selected on LB (Luria Bertani) plates containing 50 μg/mL ampicillin (Amp). Sequencing verification was performed (Qingke, Beijing, China) to obtain the engineered strains. The engineered strains were cultured at 37 °C, inoculated and expanded in LB liquid medium. Bacterial growth was monitored by measuring optical density (OD) at 600 nm using a spectrophotometer (GenStar, China).
To enhance the solubility and function of LjIFS, the N-terminus of LjIFS was modified, including truncation (removal of the first 21 amino acids) and addition of hydrophilic (KKK, HHHH) or hydrophobic tags. For CrCPR, OmpAL (localization sequence of the OmpA membrane protein) was added at the N-terminus. OmpAL acts as a signal peptide, guiding the fused exogenous protein to be transported to the cell membrane or periplasmic space via the Sec pathway.
Table 1. N-terminal sequence modifications. Underlines indicate truncated N-termini, replaced with alanine, 17A, KKK, and HHHH.
10 mM naringenin was purchased from Macklin (DMSO; N796909). The engineered bacteria were inoculated into 5 mL of LB medium (containing 50 μg/mL ampicillin) at a 1:100 ratio and cultured overnight at 37 °C. On the second day, 1 mL of the culture was transferred into 100 mL of fresh LB medium containing ampicillin. After culturing at 220 rpm and 37 °C for 12 hours, the cells were collected by centrifugation at 10,000 rpm for 1 minute. The bacterial pellet was washed once with PBS (pH 7.4). Then, the cells were transferred into 50 mL of fresh M9 medium containing 50 μg/mL ampicillin (Na₂HPO₄ 3.0 g/L, KH₂PO₄ 0.5 g/L, NaCl 1.0 g/L, NH₄Cl 1.0 g/L, MgSO₄ 5.0 mM, and CaCl₂ 0.1 mM; supplemented with 10 g/L glucose and 0.5 mM naringenin). The initial OD₆₀₀ was adjusted to 1, and fermentation was carried out at 220 rpm and 30 °C for 24 hours to produce genistein. After fermentation, 1 mL of the bacterial culture was collected and mixed with an equal volume of ethyl acetate, then vortexed for 2 hours. Centrifugation was performed at 15,000 rpm for 20 minutes, and after standing for 5 minutes, the organic phase supernatant was collected. A 200 μL aliquot of the organic supernatant was transferred into HPLC vials and evaporated to dryness in a 40 °C oven for 3–5 hours. The residue was then resuspended in 200 μL of methanol. The absorbance at 250 nm was measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific).
The cellulose synthase coding genes acsAB and acsCD were synthesized. Codon optimization for E. coli was performed, eliminating EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). Using pSB1A3 as the vector and J23100-B0034 as the promoter-RBS sequence, a polycistronic sequence was constructed. The recombinant plasmid was transformed into E. coli BL21, and positive clones were selected on LB (Luria Bertani) plates containing 50 μg/mL ampicillin (Amp) and verified by sequencing (Qingke, Beijing, China) to obtain engineered strains. The engineered strains were cultured at 37 °C, inoculated and expanded in LB liquid medium. Bacterial growth was monitored by measuring optical density (OD) at 600 nm using a microplate reader (GenStar, China).
To assay cellulose synthesis, the BL21 engineered strain was inoculated into 100 mL of fresh LB medium containing 50 μg/mL Amp. The culture was incubated at 30 °C and 180 rpm for 48 hours. After incubation, 100 mL of the bacterial culture was collected and centrifuged at 8000 rpm for 5 minutes. A 20 mL aliquot of the culture supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-cooled PBS (pH 7.4). Under ice bath conditions, the cells were disrupted using an ultrasonic homogenizer (Biosafer1000, Saifei) with settings of 150 W power, 1 s ultrasonication, 3 s intervals, for a total of 20 minutes. Next, centrifugation was performed at 15,000 × g for 20 minutes to remove cell membranes and impurities, and the supernatant was collected as the intracellular sample. To each 20 mL of intracellular and extracellular samples, 20 mL of 4 M NaOH was added and incubated at 80 °C for 2 hours to further dissolve non-cellulose components and precipitate cellulose. Then, centrifugation was performed at 15,000 × g for 30 minutes at 4 °C to collect the cellulose precipitate. The precipitated cellulose was washed repeatedly with distilled water until the pH reached 7.0 to remove residual NaOH. The washed cellulose was dried at 60 °C to constant weight. The dried cellulose was weighed to determine the cellulose content.
To assess the moisture content of the bacterial cellulose mask, the mask was folded within its packaging to ensure full absorption of the serum and allowed to soak for 30 minutes. After saturation, the wet mask was weighed to obtain the initial weight. The mask was then air-dried until no surface moisture remained and weighed again to determine the dry weight. The moisture content was calculated by subtracting the dry weight from the initial wet weight. This procedure was also performed with a commercially available mask for comparison.
To evaluate the mechanical properties of the bacterial cellulose mask, a tensile strength test was conducted. A bacterial cellulose sheet approximately 2 mm thick was prepared. A foam box filled with approximately 2 kg of water was suspended using tape attached to the bacterial cellulose sheet, which was secured at one end to a wooden stick. The setup was used to assess the mask’s ability to support weight under tension.
The blue light-inducible promoter pDawn and the red fluorescent protein mRFP were synthesized. mRFP was placed downstream of pDawn. The mRFP sequence was codon-optimized for E. coli , eliminating EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). Using pSB1A3 as the vector, pDawn-mRFP was cloned into the XbaI and SpeI restriction sites. The recombinant plasmid was transformed into E. coli DH5α, and positive clones were selected on LB (Luria Bertani) plates containing 50 μg/mL ampicillin (Amp) and verified by sequencing (Qingke, Beijing, China) to obtain the engineered strain. The engineered strain was cultured at 37 °C, inoculated and expanded in LB liquid medium. Bacterial growth was monitored by measuring optical density (OD) at 600 nm using a spectrophotometer (GenStar, China).
The blue light-inducible reporter strain was inoculated into 5 mL of LB medium containing 50 μg/mL Amp at a ratio of 1:100. Test tubes were wrapped with aluminum foil to prevent light exposure. Cultures were grown overnight in a shaking incubator at 180 rpm and 37 °C. The next day, 1 mL of bacterial culture was collected and centrifuged at 10,000 rpm for 1 minute to collect the bacterial pellet. The pellet was washed once with PBS (pH = 7.4). The cells were then transferred into 50 mL of fresh M9 medium containing 50 μg/mL ampicillin (Na2HPO4 3.0 g/L, KH2PO4 0.5 g/L, NaCl 0.5 g/L, NH4Cl 1.0 g/L, MgSO4 5.0 mM, and CaCl2 0.1 mM; supplemented with 10 g/L glucose). Five milliliters of the bacterial suspension were added to a 33 mm petri dish. The experimental group was exposed to blue light using a blue light box, while the petri dish in the control group was covered with aluminum foil. The cultures were incubated statically at 30 °C for 12 hours. Then, 200 μL of bacterial culture was taken, and fluorescence values (excitation wavelength 584 nm, emission wavelength 607 nm) and OD600 were measured using a microplate reader (Multiskan GO). The normalized fluorescence ratio (Fluorescence/OD600) was calculated.
The coding gene of the mRNA interferase mazF was inserted downstream of the pDawn promoter. The mazF sequence was codon-optimized for E. coli , eliminating EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). pDawn-mazF was cloned into the pSB1A3 plasmid (XbaI and SpeI restriction sites) and transformed into E. coli DH5α.
A 100μL of overnight culture were inoculated into 5 mL of LB medium containing ampicillin (50 μg/mL) (protected from light using aluminum foil) and cultured at 37 °C with shaking at 220 rpm for 2 hours until OD600 reached 0.4. The culture was then poured into a 33 mm petri dish and exposed to blue light (488 nm) at 30 °C. Samples of 200 μL were taken at 2 h, 4 h, 6 h, and 12 h to measure the bacterial OD600.
Data were analyzed and plotted using GraphPad Prism software. Results are presented as mean ± standard deviation (SD). For comparisons among multiple groups, one-way analysis of variance (ANOVA) and Tukey's post hoc test were used for difference analysis. For comparisons between two groups, Student's t-test was used. A p-value less than 0.05 was considered statistically significant.
