Throughout our project, we rigorously applied the Design-Build-Test-Learn (DBTL) engineering cycle to develop and optimize biological systems for heavy metal bioremediation and biosensing. Beginning with rational gene design and codon optimization, we systematically constructed and validated expression vectors for key proteins (AtPCS and PseMT) that can chelate and reduce heacy metal ions into nanoparticles in vivo, iteratively optimizing expression conditions in E. coli and ensuring robust solubility and activity. We then advanced to co-expression strategies and demonstrated functional synergy in metal tolerance and sequestration, verified both quantitatively and visually. This iterative approach extended to plant and cyanobacterial systems, where transient expression and transformation protocols were rapidly tested and refined—enabling fast cycles of hypothesis-driven experimentation. Finally, we built modular screening platforms for synthetic promoters, ready for high-throughput functional testing. At each stage, our cycles of design, construction, empirical validation, and data-driven learning enabled us to identify bottlenecks, adapt protocols, and achieve key milestones. This workflow has established a strong foundation for expanding synthetic biology applications in environmental remediation and smart biosensing.

As a seventh-year iGEM team, we are committed to the long-term impact and continuity of our project. Recognizing that several critical modules—such as stable transformation and functional demonstration in cyanobacteria, as well as the identification and validation of high-performing i-motif promoter biosensors—remain works in progress, we will carry forward these unfinished goals into iGEM 2026. Building on the strong technical foundation and engineering platforms established this year, our team will leverage the experience and data accumulated to further advance synthetic biology solutions for environmental remediation and biosensing in the next competition cycle.

1. Optimize the expressing condition of AtPCS and pseMTs in Ecoli

A key foundation of our project was the successful and efficient expression of two core proteins: phytochelatin synthase (AtPCS) from Arabidopsis thaliana and metallothionein (PseMT) from Pseudomonas in E. coli. These proteins are essential for our system’s heavy metal binding and sequestration as nano particles, so optimizing their expression was a critical early milestone.

To begin, we performed codon optimization of both AtPCS and PseMT to match E. coli’s preferred codon usage, ensuring high translation efficiency and protein yield. The optimized genes were synthesized commercially and cloned into the pET28a expression vector, which provides a strong T7 promoter for IPTG-inducible protein expression and adds a His-tag for easy detection and purification. The constructs were sequence-verified after transformation into E. coli Top10 cells for plasmid amplification, ensuring accuracy before moving to protein expression experiments.

We then transformed the validated plasmids into E. coli BL21(DE3), a strain commonly used for protein production. To systematically optimize expression, we set up a protocol where single colonies were first grown overnight in selective LB medium. The next day, cultures were diluted and grown to mid-log phase (OD₆₀₀ ≈ 0.6), then induced with a range of IPTG concentrations (from 0 to 1 mM). Importantly, we chose an induction temperature of 30°C—not only to balance protein expression and solubility, but also to simulate the environmental conditions under which our target cyanobacterium, Synechocystis sp. PCC 6803, naturally grows. Cultures were incubated at this temperature for 12 hours. After induction, we harvested the cells, lysed them under neutral conditions, and separated soluble and insoluble protein fractions by centrifugation. （details refer to： https://2025.igem.wiki/sz-shd/protocol）

The expression levels and solubility of AtPCS and PseMT were evaluated by SDS-PAGE. Distinct bands at approximately 54.5 kDa (AtPCS) and 7.9 kDa (PseMT) were visible in the soluble fractions, and the intensity of these bands increased with higher IPTG concentrations. These results confirmed that both proteins were not only highly expressed but also remained soluble under the chosen conditions—an essential requirement for their downstream metal chelation activity.

Through this systematic optimization process, we determined that inducing protein expression at 30°C with 0.5 mM IPTG for 12 hours provided the best yield and solubility for both AtPCS and PseMT. This protocol was then adopted for all subsequent co-expression and functional assays. Our work demonstrates the importance of rational gene design, careful construct validation, and systematic parameter testing—key elements of the Design-Build-Test-Learn (DBTL) cycle in synthetic biology.

2. Co-expression of AtPCS and PseMT in Ecoli

After successfully optimizing the individual expression of AtPCS and PseMT in E. coli, our next goal was to achieve their simultaneous co-expression, enabling the engineered cells to harness the synergistic heavy metal binding and detoxification properties of both proteins. To accomplish this, we constructed a dual-expression vector by assembling the codon-optimized AtPCS and PseMT genes into the same pET28a backbone using Golden Gate assembly. The resulting co-expression plasmid, pET28a-AtPCS-PseMT, was verified by nanopore whole plasmid sequencing by Genewiz.

We transformed this construct into E. coli BL21(DE3) cells, and induced protein expression with 0.5 mM IPTG at 30°C for 12 hours, following conditions previously optimized for solubility and protein yield. To evaluate whether both proteins were produced simultaneously, we extracted total soluble protein from induced and uninduced cultures and performed Western blot analysis using an anti-His tag antibody. The results revealed two distinct bands in the induced samples: one at approximately 54.5 kDa, corresponding to His-tagged AtPCS, and another at around 7.9 kDa, corresponding to His-tagged PseMT. Importantly, no such bands were observed in the negative control (cells with empty vector or uninduced cultures), confirming that the observed expression was both specific and IPTG-dependent.

The successful detection of both AtPCS and PseMT in the same sample demonstrated effective co-expression in E. coli. This achievement was further validated by the use of GAPDH as a housekeeping gene, which confirmed uniform protein loading across samples. The establishment of a robust co-expression system not only streamlines downstream functional studies but also provides a strong foundation for engineering microbial platforms capable of enhanced heavy metal remediation. By confirming that both proteins can be efficiently and specifically produced together in E. coli, we have taken a critical step forward in realizing our synthetic biology solution for environmental heavy metal pollution.

3. Function characterization of AtPCS and PseMT in Ecoli

To ensure that our assays reflected biologically meaningful stress—challenging homeostasis but not overwhelming or killing the cells outright—we grounded our metal concentration choices in published studies on E. coli metal tolerance and adaptation. For Fe²⁺, concentrations were chosen based on findings that E. coli can adapt to millimolar levels of FeSO₄, displaying both stress responses and survival (Thomas et al., 2021). For Co²⁺, we referenced Ranquet et al. 2007 (JBC) and Majtan et al. 2010, which show that cobalt at sub-millimolar to low millimolar concentrations is biologically active, disrupts iron metabolism, and induces cellular stress without causing instant lethality. For Zn²⁺ and Cu²⁺, we consulted studies demonstrating that moderate excess (typically ~0.1–0.5 mM Zn²⁺) perturbs metal homeostasis and triggers stress responses but allows for cell growth and adaptation (Osman et al., 2017, Nat Commun; Xu et al., 2019). These studies collectively justify our use of metal concentrations that are high enough to induce clear stress phenotypes and measurable biological responses, yet within the plausible range for experimental evolution and physiological challenge.

To evaluate their ability to tolerate and interact with heavy metals, we exposed E. coli strains expressing either AtPCS, PseMT, or both (PCS-MT), alongside a negative control (empty vector), to LB media supplemented with a mixture of copper (Cu), zinc (Zn), nickel (Ni), and cobalt (Co) ions at defined concentrations. After induction of protein expression with IPTG, the cultures were incubated with metals, and we monitored both cell growth and changes in medium appearance.

Strikingly, cells co-expressing AtPCS and PseMT demonstrated markedly improved tolerance to heavy metal stress compared to strains expressing either protein alone or the control. While higher concentrations of metals (5X) inhibited growth in all strains, at 1X concentration, the co-expressing strain maintained robust growth and displayed less medium discoloration, indicating reduced metal toxicity. In contrast, strains expressing only AtPCS or PseMT showed only moderate improvement over the control, underscoring the enhanced protective effect of the combined system.

To directly measure metal uptake, we quantified the residual concentrations of Cu, Zn, Ni, and Co in the culture supernatant using inductively coupled plasma optical emission spectrometry (ICP-OES) after 24 and 48 hours of incubation. The results revealed that the co-expressing strain removed approximately 50% more metal ions from the solution after 48 hours compared to the negative control, with significantly greater reduction than either single-gene strain. This confirmed that the combined action of AtPCS and PseMT confers superior metal sequestration capacity, likely due to their complementary mechanisms of chelation and binding.

Further visual confirmation of functional metal sequestration was obtained using transmission electron microscopy (TEM). Engineered E. coli cells expressing both AtPCS and PseMT accumulated dense intracellular nanoparticles following metal exposure, as evidenced by electron-dense particles within the cytoplasm. These nanoparticles were far less prevalent or absent in the control strain, providing direct ultrastructural evidence of enhanced metal accumulation by the co-expressing cells.

Taken together, these functional assays conclusively demonstrate that E. coli strains co-expressing AtPCS and PseMT exhibit much greater resilience to heavy metal stress and a higher capacity for metal uptake and nanoparticle formation than strains expressing either protein alone. This superior performance highlights the effectiveness of our synthetic biology approach and confirms that combining phytochelatin synthases with metallothioneins offers a powerful strategy for biological heavy metal remediation.

4. Expression and function validation of AtPCS and pseMTs in Moss Physcomitrella patens

To evaluate the potential of Physcomitrella patens as a chassis for heavy metal bioremediation, we focused on establishing a rapid and efficient transient expression system to test the function of two key metal-binding proteins: phytochelatin synthase (AtPCS) and Pseudomonas metallothionein (pseMT). Rather than generating stable transgenic lines—a process that is time-consuming and labor-intensive in moss—we adopted a strategy based on transient transformation, aiming for fast cycles of design, testing, and optimization. All constructs for this work were assembled in the broad-host-range pMMB67EH backbone, which supports high-level gene expression in plant systems.

Our workflow began with the preparation of sterile Physcomitrella tissue. To eliminate any background contamination, non-sterile moss from laboratory stock was transferred onto BCD medium containing 100 mg/L timentin and incubated at 25°C under continuous light. Over the course of two weeks, small fragments of moss proliferated into healthy, contaminant-free colonies, providing a reliable substrate for transformation. This step was essential to avoid microbial interference in both the transformation process and subsequent protein expression analysis.

For gene delivery, we constructed the pMMl8-35S-pcs-mt vector by cloning the codon-optimized AtPCS and pseMT sequences under the control of the cauliflower mosaic virus 35S(Cmv35s) promoter into the pMMB67EH plasmid using Golden Gate assembly. The vector was confirmed nanopore whole plasmid sequencing by genewiz, ensuring the fidelity of the assembled construct. Rather than using traditional PEG-mediated or biolistic transformation methods, we opted for a carbon nanodot-mediated transformation protocol, which allowed us to deliver plasmids efficiently into moss cells and achieve rapid, transient gene expression. In this protocol, approximately 200 mg of sterile moss tissue was incubated in a transformation solution containing MES buffer, 0.5% glycerol, and a suspension of carbon nanodots pre-complexed with the pmml8-35S-pcs-mt plasmid.(Details in https://2025.igem.wiki/sz-shd/protocol) The moss was gently agitated in this solution for twelve hours to maximize uptake, then transferred to liquid BCD medium and cultured under continuous light at 25°C.

To assess whether the introduced genes were expressed, we harvested moss samples at 0, 24, and 48 hours post-transformation and extracted total protein for western blot analysis. Membranes were probed with an anti-His tag antibody to detect the recombinant AtPCS (~54.5 kDa) and pseMT (~7.9 kDa) proteins, and then reprobed with anti-plant β-actin antibody as a loading control. Robust expression of both AtPCS and pseMT was observed at 24 and 48 hours, with negligible signal at 0 hours, confirming the effectiveness of the transient transformation system and the activity of our pMMB67EH-based construct in Physcomitrella cells.

Having established successful protein expression, we next sought to determine whether these recombinant proteins conferred enhanced heavy metal uptake and nanoparticle biosynthesis. Transformed moss tissues were exposed to solutions containing copper, zinc, nickel, and cobalt ions for 24–48 hours. Following metal exposure, tissues were fixed with glutaraldehyde and prepared for analysis by environmental scanning electron microscopy (ESEM). ESEM imaging revealed clear aggregates of electron-dense metal nanoparticles distributed across the surface of the moss at both 1,000× and 2,500× magnification, as indicated by red arrows in the micrographs. These aggregates were not present in untransformed control moss, demonstrating that transiently expressed AtPCS and pseMT enabled the moss to synthesize and accumulate metal nanoparticles extracellularly under metal stress.

5. Expression of AtPCS and pseMTs in Cyanobacteria PCC6803 and integration into bioreactor

To further expand the versatility and sustainability of our heavy metal bioremediation platform, we sought to harness the unique advantages of the cyanobacterium Synechocystis sp. PCC6803. This photosynthetic microorganism is attractive for environmental applications due to its ability to grow autotrophically under light, its ease of cultivation in aqueous systems, and its suitability for scale-up in controlled bioreactors. Our goal was to engineer PCC6803 to co-express Arabidopsis phytochelatin synthase (AtPCS) and Pseudomonas metallothionein (pseMTs), and to lay the groundwork for future bioprocess development.

The engineering process began with the design and assembly of an expression vector tailored for use in cyanobacteria. We first validated the function of a theophylline-inducible promoter system by constructing a reporter plasmid expressing blue fluorescent protein (BFP), using BBa_K592100 from the iGEM 2023 distribution kit. After confirming that the promoter permitted gene induction in Synechocystis cells, we replaced the BFP reporter with codon-optimized pcs and mt genes, enabling potential co-expression of AtPCS and pseMTs in response to theophylline. The use of the pMMB67EH backbone facilitated modular cloning and broad-host-range compatibility, supporting our synthetic biology approach.

Transformation of cyanobacteria presents unique challenges, particularly with respect to efficiency and stability. We employed the natural transformation protocol for Synechocystis PCC6803, leveraging its innate competence. Exponentially growing cells were incubated with our constructed vector and plated on BG11 agar containing kanamycin as a selective agent. Despite repeated attempts, we observed no colony formation on plates containing the transformed cells, while control plates also showed no growth, confirming the stringency of selection. The lack of transformants suggested that our protocol may require further optimization or that alternative transformation strategies—such as conjugation or electroporation—may be needed to achieve successful integration and expression of the target genes.

Although we were not able to generate engineered PCC6803 strains within this cycle, we proceeded to characterize the growth and biomass production of wild-type cultures in a laboratory bioreactor as a foundation for future system integration.

Cultures were grown at 30°C under continuous illumination of 5000 LUX, and OD600 was measured daily to monitor cell density. The growth curve closely followed the Gompertz model, with an excellent fit (R² = 0.9905), providing a robust quantitative framework for predicting culture performance and planning scale-up .

In parallel, we measured the wet biomass yield by harvesting 10 mL culture samples over three days, with three replicates per day. The average biomass yield was determined to be 0.0087 g per OD per mL of medium, a key metric for future assessments of heavy metal uptake and process throughput .

Overall, while stable expression of AtPCS and pseMTs in Synechocystis PCC6803 remains an ongoing challenge, our work has established the molecular tools and bioreactor framework necessary for the next stage of synthetic biology-driven bioremediation. The validation of the inducible promoter system and the baseline bioreactor data provide a strong platform for iterative optimization. In future cycles, we aim to improve transformation efficiency, explore alternative gene delivery methods, and ultimately demonstrate the light-driven, scalable removal of heavy metals from aqueous environments using engineered cyanobacteria. This integrated approach will advance the realization of sustainable, biotechnological solutions for environmental remediation.

6. Engineering a Screening Platform for Carbon Dot-Inducible Promoters as Sensors of Cellular Saturation with Reduced Metal Nanoparticles

As the field of plant synthetic biology advances toward increasingly precise and responsive genetic circuits, the need for biosensors that can report on cellular status in real time becomes ever more pressing. In response, we have engineered a modular screening system to develop synthetic promoters that can indicate when a plant cell is saturated with reduced metal nanoparticles—a common endpoint in phytoremediation and plant nanobiotechnology.

Building on previous discoveries that carboxyl-modified carbon dots can modestly activate native i-motif-containing promoters in rice, we hypothesized that synthetic promoters incorporating these motifs could serve as sensitive elements for detecting changes in intracellular nanoparticle concentrations.

To enable rapid functional screening, we constructed a robust dual-reporter platform based on the pGreen-mscarlet-lv0 vector, which includes both luciferase and fluorescent reporters, alongside a Golden Gate-compatible entry system. This modular setup allows us to efficiently clone individual i-motif sequences upstream of a minimal promoter and quantitatively assess their responsiveness within plant cells.

With this testing system in place, our next step is to systematically screen the library of i-motif sequences by introducing them into the reporter construct and evaluating their activation in response to carbon dot exposure and intracellular accumulation of reduced metal nanoparticles. By measuring luciferase activity and fluorescence output, we aim to identify specific i-motif configurations that act as effective sensors for when the cell approaches or reaches saturation with metal nanoparticles.

Although the optimal i-motif-based promoter has not yet been identified, the establishment of this screening platform represents a significant engineering milestone. It provides a direct and scalable route to discovering new biosensors that can be integrated into regulatory circuits for plant-based nanomaterial production, environmental monitoring, or phytoremediation. Once validated, such promoters could enable plants to autonomously report on their nanoparticle load, paving the way for feedback-regulated expression systems and safer, more efficient metallophyte applications.

References:

• Majtan, T., Frerman, F. E., & Kraus, J. P. (2011). Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation. Biometals, 24(2), 335–347. https://doi.org/10.1007/s10534-010-9400-7
• Osman, D., Foster, A. W., Chen, J., Svedaite, K., Steed, J. W., Lurie-Luke, E., Huggins, T. G., & Robinson, N. J. (2017). Fine control of metal concentrations is necessary for cells to discern zinc from cobalt. Nature Communications, 8(1), Article 1884. https://doi.org/10.1038/s41467-017-02085-z
• Ranquet, C., Ollagnier-de-Choudens, S., Loiseau, L., Barras, F., & Fontecave, M. (2007). Cobalt stress in Escherichia coli: The effect on the iron-sulfur proteins. The Journal of Biological Chemistry, 282(42), 30442–30451. https://doi.org/10.1074/jbc.M702519200
• Thomas, M. D., Ewunkem, A. J., Boyd, S., Williams, D. K., Moore, A., Rhinehardt, K. L., Van Beveren, E., Yang, B., Tapia, A., Han, J., Harrison, S. H., & Graves, J. L., Jr. (2021). Too much of a good thing: Adaption to iron (II) intoxication in Escherichia coli. Evolution, Medicine, and Public Health, 9(1), 53–67. https://doi.org/10.1093/emph/eoaa051
• Xu, Z., Wang, P., Wang, H., Yu, Z. H., Au-Yeung, H. Y., Hirayama, T., Sun, H., & Yan, A. (2019). Zinc excess increases cellular demand for iron and decreases tolerance to copper in Escherichia coli. The Journal of Biological Chemistry, 294(45), 16978–16991. https://doi.org/10.1074/jbc.RA119.010023