1. Successful transformation of pET28a-AtPCS and Pet28a-PseMT plasmid into Ecoli BL21(DE3)
The AtPCS and pseMT gene were codon optimized, synthesised and coloned into the pET28a plasmid by Tsingke biotech.
Plasmid map for pET28a-AtPCS
Plasmid map for pET28a-pseMT
Once the plasmid were received, it was then transformed into Top10 competent cell for amplification and Ecoli BL21(DE3) competent cell for downstream test. The single colony was verified by whole nanopore sequencing by Genewiz biotech.
2.Construct of pET28a-AtPCS-pseMT co expressing vector by golden gate
The AtPCS and pseMT gene were codon optimized, synthesised and coloned into the pET28a plasmid by Tsingke biotech.
3.Optimize expression condition of AtPCS and PseMT in Ecoli BL21(DE3)
To optimize the expression of AtPCS protein in E. coli BL21(DE3), cultures were induced with increasing concentrations of IPTG (0–1 mM) at 30°C for 12 hours. After induction, cells were harvested and lysed under neutral conditions, and the soluble protein fractions were collected by centrifugation. The soluble proteins were analyzed by SDS-PAGE. A distinct band at approximately 54.5 kDa, corresponding to the expected size of AtPCS, was observed in samples induced with IPTG. The intensity of this band increased with higher IPTG concentrations, indicating enhanced expression and solubility of AtPCS under these conditions.
SDS-PAGE analysis of AtPCS expression in E. coli BL21 (DE3) under different IPTG induction concentrations at 30°C for 12 hours. Lane 1: protein marker; Lane 2: 0 mM IPTG; Lane 3: 0.2 mM IPTG; Lane 4: 0.4 mM IPTG; Lane 5: 0.6 mM IPTG; Lane 6: 0.8 mM IPTG; Lane 7: 1 mM IPTG. The arrow indicates the induced AtPCS protein band at ~55 kDa.
To determine the optimal induction conditions for soluble expression of PseMT in E. coli BL21(DE3), cells were induced with increasing concentrations of IPTG (0–1 mM) at 30°C for 12 hours. After induction, cells were harvested and lysed under neutral conditions, and the soluble protein fraction was collected by centrifugation. SDS-PAGE analysis of the soluble proteins revealed a clear band at approximately 7.9 kDa, corresponding to the expected molecular weight of PseMT, in samples induced with IPTG. The band intensity increased with higher IPTG concentrations, indicating that PseMT is efficiently expressed and remains soluble under these conditions.
SDS-PAGE analysis of soluble PseMT protein expressed in E. coli BL21(DE3) following induction with increasing concentrations of IPTG at 30°C for 12 hours. After cell lysis under neutral conditions, only the soluble protein fractions were analyzed. Lane 1: protein marker; Lane 2: 0 mM IPTG; Lane 3: 0.2 mM IPTG; Lane 4: 0.4 mM IPTG; Lane 5: 0.6 mM IPTG; Lane 6: 0.8 mM IPTG; Lane 7: 1 mM IPTG. The arrow indicates the soluble PseMT protein band (~10 kDa).
4.Verify the co-expression of AtPCS and PseMT in Ecoli
To confirm the co-expression of AtPCS and PseMT proteins in E. coli BL21(DE3), cells with pET28a-pcs-mt plasmid were induced with 0.5 mM IPTG at 30°C for 12 hours. Total soluble proteins were extracted and analyzed by Western blot using an anti-His tag antibody. The results show distinct bands at approximately 55 kDa and 10 kDa, corresponding to His-tagged AtPCS and PseMT, respectively, in the induced sample. No such bands were detected in the negative control (nc, uninduced cells), confirming successful and specific co-expression of both target proteins. GAPDH was used as a loading control and incubated after stripping the primary antibody.
Western blot analysis confirming co-expression of His-tagged AtPCS (~55 kDa) and PseMT (~10 kDa) in E. coli BL21(DE3). Lane 1: protein molecular weight marker; Lane 2: negative control (nc, uninduced); Lane 3: cells induced with 0.5 mM IPTG. Blots were probed with an anti-His tag antibody. Bands corresponding to AtPCS and PseMT are visible only in the induced sample, indicating successful co-expression of both proteins. GAPDH served as a loading control.
5.Verify the function of AtPCS and PseMT in Ecoli
The Ecoli cell was first incubated to od600≈0.6 at 37°C with shaking at 220 rpm， . 0.5mM IPTG were added into the medium and incubate at 30°C with shaking at 220 rpm for 4hr.
Then 1.5 mM Cu, 2.5 mM Zn, 3 mM Ni, and 1.5 mM Co ion were added into the LB medium and incubate at 30°C with shaking at 220 rpm for 24hr. The Color of the medium was been observed.
Escherichia coli cells were incubated in LB medium supplemented with 1.5 mM Cu, 2.5 mM Zn, 3 mM Ni, and 1.5 mM Co at 30°C with shaking at 220 rpm for 24 hours. NC: cells with the empty pET28a vector; pseMTs: cells expressingpseMT; AtPCS: cells expressing AtPCS.
6.Verify the function of co expressing AtPCS and PseMT in Ecoli
To verify the function of the PCS-MT co-expressing construct in E. coli, cells contain either the empty pET28a vector (NC) or the pET28a-PCS-MT vector were first grown to an OD600 ≈ 0.6 at 37°C with shaking at 220 rpm. Protein expression was induced with 0.5 mM IPTG, followed by incubation at 30°C with shaking at 220 rpm for 4 hours.
Cells were then transferred into LB medium supplemented with either 1X (1.5 mM Cu, 2.5 mM Zn, 3 mM Ni, and 1.5 mM Co) or 5X (7.5 mM Cu, 12.5 mM Zn, 15 mM Ni, and 7.5 mM Co) concentrations of metal ions, and incubated at 30°C with shaking at 220 rpm. Samples were taken at 0, 24, and 48 hours to observe changes in cell growth and medium color due to the synthesis of metal nano dots. As shown in the figure, growth was inhibited at higher metal concentrations (5X), while cells expressing the PCS-MT construct at 1X concentration showed better growth compared to the negative control.
Growth of E. coli expressing the pET28a-PCS-MT co-expression vector under different metal concentrations over time. NC: negative control with empty pET28a vector. 1X: pET28a-PCS-MT with 1.5 mM Cu, 2.5 mM Zn, 3 mM Ni, and 1.5 mM Co. 5X: pET28a-PCS-MT with five times the 1X metal concentrations. Cultures were incubated at 30°C and sampled at 0, 24, and 48 hours.
After 24 hours of incubation in LB medium supplemented with 1.5 mM Cu, 2.5 mM Zn, 3 mM Ni, and 1.5 mM Co at 30°C with shaking at 220 rpm, E. coli cells were harvested by centrifugation at 8000 × g for 1 minute. The cell pellet from the control (NC, empty pET28a vector) appeared off-white, while the pellet from cells expressing the pET28a-PCS-MT co-expression vector (1X) exhibited a distinct brown coloration. This color change indicates the biosynthesis of metal nanodots by the engineered E. coli.
Cell pellets of E. coli after 24-hour incubation in LB medium containing 1.5 mM Cu, 2.5 mM Zn, 3 mM Ni, and 1.5 mM Co at 30°C 24hr, followed by centrifugation at 8000 × g for 1 minute. NC: cells with the empty pET28a vector; 1X: cells expressing the pET28a-PCS-MT co-expression vector. The brown coloration of the 1X pellet indicates the synthesis of metal nanodots by the engineered cells.
7.Verify the function of co expressing AtPCS and PseMT in Ecoli through Mass spectrometer
After incubation of E. coli cultures with metal ions, the concentrations of residual copper (Cu), zinc (Zn), nickel (Ni), and cobalt (Co) in the culture supernatant were determined using inductively coupled plasma mass spectrometry (ICP-MS). At each time point (24 and 48 hours), the cultures were centrifuged at 8000 × g for 1 minute to pellet the cells, and the supernatant was carefully collected to avoid disturbing the pellet. The collected supernatant samples were diluted tenfold with ultrapure water to ensure the metal concentrations fell within the linear detection range of the instrument.
The ICP-OES was operated under the following conditions: RF power was set to 1.20 kW, with a plasma gas (argon) flow of 15.0 L/min and an auxiliary gas flow of 1.50 L/min. The nebulizer flow rate was maintained at 0.75 L/min. For each sample, a sample uptake delay of 15 seconds was used to ensure accurate introduction of the sample into the plasma. After the uptake period, the instrument stabilization delay was set to 15 seconds to ensure stable signal acquisition. Each measurement was performed in triplicate, with a replicate read time of 2 seconds.
Workflow for sample preparation and analysis of metal ion concentration by ICP-OES.(1) Preparation of reagents and dilution of culture supernatant samples under a fume hood.(2) Aliquoting and organization of diluted samples into tubes for analysis.(3) Measurement of metal ion concentrations using an Agilent 720ES inductively coupled plasma optical emission spectrometer (ICP-OES).
Quantification was based on calibration curves generated from certified standard solutions for each metal. The raw concentration readings (mg/L) were multiplied by the dilution factor to calculate the final concentration in the original samples. The average of three technical replicates was reported for each sample.
Concentration of Cu, Zn, Ni, and Co ions remaining in the culture supernatant as measured by mass spectrometry (mg/L). Orange bars indicate control samples (without plasmid), gray bars indicate samples with the pET28a-PCS-MT co-expression plasmid after 24 hours, and blue bars indicate samples with the plasmid after 48 hours incubation. A significant reduction(~50%) in metal ion concentration was observed in cultures expressing the plasmid, particularly after 48 hours, demonstrating the enhanced metal uptake by engineered E. coli.
8.TEM detection of metal nano particle
Transmission electron microscopy (TEM) was used to visualize intracellular metal nanoparticles in engineered E. coli cells. Cells were fixed with 2.5% glutaraldehyde fixing solution and sent to lab using ice pack. Once arrival, it was post-fixed with osmium acid, washed, dehydrated through an ethanol series, and embedded in a resin-acetone mixture. Ultrathin sections (70–90 nm) were prepared using a UC7 Ultramicrotome (Leica, Germany), then stained with uranyl acetate and lead citrate. Samples were imaged on a JE 1200-EX TEM (JEOL, Japan). The resulting micrographs revealed distinct, electron-dense nanoparticles (indicated by red arrows) within the cytoplasm of E. coli, confirming the biosynthesis and intracellular accumulation of metal nanoparticles.
TEM image of engineered E. coli cells showing intracellular electron-dense metal nanoparticles (red arrows). Cells were processed according to standard TEM protocols, including fixation with glutaraldehyde and osmium acid, dehydration, resin embedding, ultrathin sectioning (70–90 nm), and staining with uranyl acetate and lead citrate. Imaging was performed on a JE 1200-EX TEM (JEOL, Tokyo, Japan).
9.Separation of sterile strain Moss Physcomitrella patens
Non-sterile Physcomitrella patens moss from our laboratory stock was cultured on BCD medium supplemented with 100 mg/L timentin to eliminate potential microbial contaminants and obtain sterile plants. Moss fragments were transferred to the selective medium and incubated at 25°C under continuous light (5000 LUX, 24 hours). On Day 0, small pieces of moss were placed onto the medium. By Day 14, healthy and contaminant-free moss colonies were observed, demonstrating successful sterilization and propagation. This protocol enables the establishment of sterile Physcomitrella patens cultures for further genetic and physiological studies.
Establishment of sterile Physcomitrella patens cultures from non-sterile laboratory stock. Moss was transferred to BCD medium containing 100 mg/L timentin and incubated at 25°C with 5000 LUX continuous light. Left: Moss fragments at Day 0. Right: Proliferation of sterile moss after 14 days.
10.Construction of moss expression vector pmml8-35s-pcs-mt using golden gate assembly
11.Transformation of Moss Physcomitrella patens
To generate transient expression Physcomitrella patens expressing phytochelatin synthase (AtPCS) and Pseudomonas metallothioneins (pseMTs), moss tissue was transformed using carbon nano dots as carriers. Approximately 200 mg of moss was incubated in transformation solution containing MES buffer (pH 5.5), 0.5% glycerol, and endotoxin-free transformation vector for 12 hours. Following transformation, moss was cultured in liquid BCD medium at 25°C under continuous light for 24 hours. Protein expression was evaluated at 0, 24, and 48 hours post-transformation using western blotting. Membranes were first probed with anti-His tag antibodies to detect recombinant AtPCS and pseMTs, then stripped and reprobed with anti-β-actin antibody as a loading control. Robust expression of both AtPCS (~54.5 kDa) and pseMTs (~7.9 kDa) was observed at 24 and 48 hours, confirming successful transformation and expression in the moss system.
Western blot analysis of AtPCS and pseMTs expression in Physcomitrella patens following transformation with carbon nano dots. Total protein was extracted at 0, 24, and 48 hours post-transformation. Blots were probed with anti-His tag antibody to detect AtPCS (~54.5 kDa) and pseMTs (~7.9 kDa), then reprobed with anti-β-actin antibody as internal reference (~39 kDa). Strong bands at 24 and 48 hours indicate successful uptake and expression of both recombinant proteins in moss tissue.
12.Detecction of metal nano particle synthesized by moss using SEM
To confirm the biosynthesis of metal nanoparticles by Physcomitrella patens, moss tissues were fixed in 2.5% glutaraldehyde and analyzed using an environmental scanning field electron microscope (ESEM, Quattro). Imaging was performed at an accelerating voltage of 5.00 kV, a beam current of 57 pA, and working distance of 5.4 mm. Secondary electron detection (ETD, SE mode) was used to visualize surface features. The resulting images show clear aggregates of nanoparticles (indicated by red arrows) distributed on the moss tissue surface at both 1,000× and 2,500× magnification. These observations confirm the ability of moss to synthesize and accumulate metal nanoparticles extracellularly under the tested conditions.
Environmental scanning electron microscope (ESEM) images of Physcomitrella patens tissue after metal nanoparticle biosynthesis. Moss samples were fixed with 2.5% glutaraldehyde and imaged using a Quattro ESEM at 5.00 kV accelerating voltage, 57 pA beam current, 5.4 mm working distance, and secondary electron detection (SE mode). Nanoparticle aggregates (red arrows) are visible on the moss surface at 1,000× magnification (left, scale bar: 40 μm) and 2,500× magnification (right, scale bar: 10 μm), confirming successful nanoparticle formation.
13.Construction of cyanobacteria expression vector
To verify the function of the theophylline induced promoter in cyanobacteria, we constructed a expressing vector contain theophylline induced promoter and report gene BFP BBa_K592100 in igem 2023 distribution kit.
To express pcs and mt in cyanobacteria pcc6803, we replace the BFP gene with pcs and mt gene.
14.Nature transformation of Cyanobacteria pcc6803
Then, we transform the Cyanobacteria pcc6803 using the nature transformation method(https://2025.igem.wiki/sz-shd/protocol) and plate the cell on BG11 plate contain kanamycin. Sadly, no colony were formed after several times of trail.
Natural transformation of Synechocystis sp. PCC 6803. Left: Control BG11 plate containing kanamycin shows no growth. Right: BG11 plate with transformed cells also shows an absence of colony formation after incubation, indicating that no kanamycin-resistant transformants were obtained.
15.Construction of light bio reactor
We grow the wildtype pcc6803 in bio reactor with 30℃ 5000LUX 24hr light for and measure the od600 value every day.
Growth curve of wild-type Synechocystis sp. PCC 6803 in a light bioreactor at 30°C with continuous 5000 LUX illumination. Blue dots represent observed OD600 values measured daily. The orange solid line shows the Gompertz model fit (R² = 0.9905), and the yellow dashed line represents the model extension beyond the observation period. The fitted equation and parameters are indicated at the bottom of the plot.
16.Measurement of PCC6803 biomass in bioreactor
We measure the weight of pcc6803 in 10ml medium in our bioreactor for 3 different days and 3 times each day. The average is 0.0037g/od/ml medium.
Measurement of wet biomass for Synechocystis sp. PCC 6803 cultures from the bioreactor. Representative image showing a pre-weighed tube containing harvested cyanobacterial cells. Biomass was measured from 10 mL culture samples across three days (three replicates per day). The average yield was determined to be 0.0087 g/OD/mL medium.