iGEM 2025 — Team hkust

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![Title](https://static.igem.wiki/teams/5546/title/10-1.webp)

|  |
| --- |
| Notes |
| [Abbreviations](https://static.igem.wiki/teams/5546/notebook/abbreviations.pdf) |
| June |
| [Week 1](https://static.igem.wiki/teams/5546/notebook/updatedweek1-compressed.pdf) |
| [Week 2](https://static.igem.wiki/teams/5546/notebook/updatedweek2-compressed.pdf) |
| [Week 3](https://static.igem.wiki/teams/5546/notebook/updatedweek3-compressed.pdf) |
| [Week 4](https://static.igem.wiki/teams/5546/notebook/updatedweek4-compressed.pdf) |
| [Week 5](https://static.igem.wiki/teams/5546/notebook/updatedweek5-compressed.pdf) |
| July |
| [Week 6](https://static.igem.wiki/teams/5546/notebook/updatedweek6-compressed.pdf) |
| [Week 7](https://static.igem.wiki/teams/5546/notebook/updatedweek7-compressed.pdf) |
| [Week 8](https://static.igem.wiki/teams/5546/notebook/updatedweek8-compressed.pdf) |
| [Week 9](https://static.igem.wiki/teams/5546/notebook/updatedweek9-compressed.pdf) |
| August |
| [Week 10](https://static.igem.wiki/teams/5546/notebook/updatedweek10-compressed.pdf) |
| [Week 11](https://static.igem.wiki/teams/5546/notebook/updatedweek11-compressed.pdf) |
| [Week 12](https://static.igem.wiki/teams/5546/notebook/updatedweek12-compressed.pdf) |
| [Week 13](https://static.igem.wiki/teams/5546/notebook/updatedweek13-compressed.pdf) |
| September |
| [Week 14](https://static.igem.wiki/teams/5546/notebook/updatedweek14-compressed.pdf) |
| [Week 15](https://static.igem.wiki/teams/5546/notebook/updatedweek15-compressed.pdf) |
| [Week 16](https://static.igem.wiki/teams/5546/notebook/updatedweek16-compressed.pdf) |
| [Week 17](https://static.igem.wiki/teams/5546/notebook/updatedweek17-compressed.pdf) |
| [Week 18](https://static.igem.wiki/teams/5546/notebook/updatedweek18-compressed.pdf) |

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* [Overview](#overview)
* [Engineering Success](#engineering-success)
* [Cycle 0: Ideation](#cycle-0)
* [Cycle 1: Metallothionein in *E. coli*](#cycle-1)
* [Cycle 2: Metallothionein in *P. putida*](#cycle-2)
* [Cycle 3: Biosensor](#cycle-3)
* [Cycle 4: Kill Switch](#cycle-4)
* [Future Cycle](#future-cycle)

☰ Menu

![Title](https://static.igem.wiki/teams/5546/title/9-1.webp)

HKUST iGEM 2025 Team Slupeer is committed to addressing heavy metal pollution through an innovative, eco-friendly approach using bio-engineered bacteria for cadmium extraction. Guided by iGEM’s core engineering principles - design, build, test, and learn - our team has iteratively refined the project through multiple engineering cycles to achieve its current optimized form.

## Engineering Success

---

Through a series of Engineering cycles, Team Slupeer successfully constructed the plasmid for the expression of Lpp-OmA-Metallothionein ([BBa\_25OWO9RF](https://registry.igem.org/parts/bba-25owo9rf)) via Gibson assembly and transformed it in both *E. coli* BL21 Rosetta and *P. putida* KT2440 (please refer to the [Cloning l Protocols](https://2025.igem.wiki/hkust/protocols) and [Experiments](https://2025.igem.wiki/hkust/experiments) for more details). Upon successful transformation, 4 main experiments – cell viability assay, Western blot, TEV protease cleavage test, and ICP-OES – were performed, which confirmed the expression and functionality of the metallothionein, PflQ2MT, and showed high potential of using bio-engineered *P. putida* for cadmium extracting bioremediation.

![Figure 1](https://static.igem.wiki/teams/5546/engineering/fig11.avif)

*Figure 1.* Bar chart of OD600 of EC and PP at 30 µM Cd2+. Student’s t-test ∗∗P \< 0.01, ∗∗∗P \< 0.001, n \= 3.

Figure 1 shows that *E. coli* with MT (ECMT) has a higher OD600 than *E. coli* (EC), which justifies that MT does increase the survival of bacteria in cadmium solutions. Furthermore, at 30 µM Cd2+, *P. putida* has a significantly larger OD600 than *E. coli*. Hence, it is concluded that the metallothionein protein increases the survivability of the bacteria, and *P. putida* is a more advantageous candidate as a chassis to remove cadmium.

![Figure 2](https://static.igem.wiki/teams/5546/engineering/fig12.avif)

*Figure 2.* ICP-OES result of *E. coli* with different treatments in a 60 µM Cd2+ solution. n \= 2-3.

Moreover, we utilized ICP-OES to detect the cadmium concentrations after the solutions were incubated with ECMT overnight. As seen in Figure 2, the normalised value of the change in Cd2+ concentration to the OD showed that E. coli with MT causes a higher decrease in cadmium concentration, which proposes its potential to bind Cd2+ and extract it from the solutions. The data further affirms the engineering success of the basic part ([BBa\_25VCEOE5](https://registry.igem.org/parts/bba-25vceoe5)), and evidence supporting its binding function.

While our [Experiments](https://2025.igem.wiki/hkust/experiments) page documents a range of data with potential insights and promise, we believe these two figures most strongly demonstrate our success in achieving the goal of our original engineering success. To note, the goal of Engineering Cycle 0 was to produce Wet Lab data that affirms in-silico work surrounding the function of PflQ2MT as a potential bioremediating protein candidate. Subsequently, Engineering Cycle 1 aimed to utilise a more characterised system in E. coli to test the function of the MT circuit and confirm the subsequent added functions compared to the wild type strain. From our results, we believe these figures demonstrate strong engineering success and show we have managed to accomplish and undergo the two original goals set for these engineering cycles.

If you would like to know more about how we evaluated our Engineering Success for real-world implemntation, please take a look at our[Application Development](https://2025.igem.wiki/hkust/human-practices#application-development) Page.

## Cycle 0

---

Our journey of engineering began with an in-depth literature review and discussions to identify effective metal-binding proteins for heavy metal extraction. Continuous engagement with field experts provided critical feedback, shaping the project’s direction and enhancing the design and functionality of our system.

Cycle 0: Ideation
Expand All

Design

The primary focus of the first design part was to narrow down our project’s scope. So to better understand cadmium pollution and determine cadmium as our dedicated primary goal, we conducted an interview with Dr. Xiyue JIA, who was an expert in cadmium contamination in rice.

Next, as we found EC20 and metallothionein (MT) to be strong candidates with their capabilities of binding to heavy metal ions, hence we also reached out to Dr. Weon Bae, the first author of multiple EC20-related articles to gain more insights. To learn more about the interviews, refer to [integrated Human Practice](https://2025.igem.wiki/hkust/human-practices) page.

Drawing inspiration from successful heavy metal sequestration strategies documented within the iGEM Registry, we determined that surface display was the optimal approach compared to intracellular binding. This choice offers significant advantages, such as enhanced accessibility for binding target ions, simplified downstream separation of the biosorbent, and potential for regeneration and reuse. To achieve robust surface binding, we screened various membrane localization systems and ultimately selected a membrane fusion protein system designed for anchoring the binding protein onto the cell membrane. We eventually selected the well-documented Lpp-OmpA hybrid system. In this construct, the Lpp (Lipoprotein) domain serves as the membrane anchor, ensuring stable localization to the outer membrane, while the OmpA (Outer Membrane Protein A) domain functions as the linker protein, providing a stable scaffold for displaying the target binding domain on the cell surface ((Higham et al., 1985; INP-NC, n.d.; Krogsgaard et al., 2000; Narita et al., 2005; Parks et al., 2024)).

![Figure 3](https://static.igem.wiki/teams/5546/engineering/fig1.avif)

*Figure 3.* The optimal selection process of our anchor protein. From general meeting on 25th Apr.

Build

As part of our iHP work, we developed our own criteria on the ideal metal-binding protein which involved:

1. Specificity
2. Sensitivity
3. Binding Efficiency

In order to choose the best binding protein, instead of relying solely on Human Practices, we also wanted to screen through the proteins ourselves. Hence, we build a computational workflow to narrow down potential candidates and to try to predict the potential properties of said candidates.

To investigate the structural properties of EC20 and other MTs, phytochelatins, and functionally homologous proteins for cadmium binding, we utilized AlphaFold 3 to predict their folding, both in isolation and in complex with the Lpp-OmpA delivery system. The process of building the workflow and subsequent analysis is described thoroughly in the “MT Cycle 0” under our [Model](https://2025.igem.wiki/hkust/model) Page. As a general summary, it involved building a library of different parts from existing literature and the iGEM Registry and analysing pLDDT and pTM scores to assess the reliability of predicted structures. Candidates were first folded in isolation to ensure pLDDT values indicated medium to high confidence before proceeding with Lpp-OmpA folding simulations. Zinc ions, as cadmium analog, were used as a proxy in AlphaFold 3 simulations, given their chemical and structural similarities and documented affinity of MTs for both metals. As metal-binding proteins have dynamic structures, the presence of ions is crucial to provide context in the folding of the protein. Structural stability and binding potential were evaluated using pLDDT (>70 for reliable conformations), pTM (>0.6 for overall model quality), and ipTM scores (for protein-metal complex stability). This approach enabled the identification of well-defined protein structures with potential for effective cadmium binding under physiological conditions, providing a robust framework for further development of metal-binding proteins.

Test

Due to limitations in AlphaFold 3 for predicting Cd²⁺ binding, we employed the MIB2 server to identify and model metal ion-binding sites in PflQ2MT, evaluating 18 metal ions with focuses on ions which are more commonly found in sludge or may compete with Cd². This included Cu²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Zn²⁺, Cd²⁺, Fe²⁺, Ni²⁺, Hg²⁺, Co²⁺ and Pb²⁺. applications.

Due to various reasons, which were outlined in our [Model](https://2025.igem.wiki/hkust/model) page, we switched from EC20 to PflQ2MT. Our analysis of PflQ2MT revealed that up to 9 cysteine residues participate in binding, as determined by docking scores and structural analysis using MIB2. Several metal ions, including Cu²⁺, Fe³⁺, Zn²⁺, Hg²⁺, Co²⁺, and Au²⁺, showed affinity for individual cysteine residues, but only Mn²⁺, Cd²⁺, Fe²⁺, Ni²⁺, Cu⁺, Ba²⁺, and Pb²⁺ effectively bind within the protein’s three-dimensional structure. PflQ2MT exhibited the greatest preference for Cd²⁺ and Cu⁺, each binding to all 9 cysteines, followed by Fe²⁺ binding to 7 cysteines and Pb²⁺ to 5 cysteines. Although Cu⁺ binds effectively, its instability and tendency to oxidize to Cu²⁺, which does not bind any cysteines, makes it less competitive than Cd²⁺. Consequently, Cd²⁺ is the most preferentially bound metal ion by PflQ2MT due to its strong interaction with all 9 cysteines and minimal competition from Cu²⁺.

Learn

Our Dry Lab outputs highlighted the limitations of utilising prediction tools with static outputs such as the MIB2 algorithm and AlphaFold 3. Particularly, its application in predicting cadmium ion binding to dynamic proteins like LgiMT1 and LgiMT2 highlight the need for more robust computational approaches. MIB2's treatment of proteins as static structures fails to account for significant topological changes during metal ion binding, leading to inaccurate predictions of binding sites and affinities. Thus, this shifted our paradigm in future cycles.

1. To improve accuracy, our later engineering cycles document our work in developing molecular dynamics simulations.
2. We aimed to avoid developing fully in-silico models, instead using wet lab data and existing literature to ensure reliable outputs.

Our dry lab screening [results](https://2025.igem.wiki/hkust/model) demonstrated that the PflQ2MT metallothionein is superior to EC20 in several key aspects. Specifically, PflQ2MT offers a better burden-effectiveness ratio, suggesting optimal performance relative to the metabolic load it imposes on the host cell. Furthermore, the gene encoding PflQ2MT is structurally simpler, featuring fewer repetitive sequences, which facilitates easier and more reliable synthesis and cloning. This selection aligns with our overall strategy of utilizing surface expression, which is inherently advantageous because it minimizes the metabolic burden placed upon the cell compared to high-level intracellular expression. By localizing the binding protein externally, we ensure that the host cell's internal machinery remains largely dedicated to growth and viability, optimizing overall biosorbent production efficiency.

Our focus on heavy metal remediation is crucial because cadmium is recognized as a serious and pervasive environmental pollutant. As a highly toxic heavy metal, cadmium poses significant risks to human health and ecosystems, necessitating the development of efficient and sustainable bioremediation solutions. In the ideation stage of our project development, we found challenges in project positioning and developing an optimal application model. As a result, we sought stakeholders from multiple fields including heavy metal pollution, cadmium and sludge treatment to better fit our project into the demands of the industry. The detailed process and feedback documentation can be found in our [Application Development page](https://2025.igem.wiki/hkust/human-practices#application-development).

## Cycle 1

---

From our research, we found that many parts we wanted to test were more well-characterised in *E. coli*, making it a better-documented and more appropriate system to initial our design and experiments. Moreover, we need to start constructing our plasmid from *E. coli* for easier transformation and selection.

Cycle 1: Metallothionein in *E. coli*
Expand All

Design

The circuit we designed for expressing MT in *E.coli* contains 3 main parts, which are anchor protein fused with a protease cleavage site, downstreaming the cadmium binding protein PflQ2MT ([BBa\_25VCEOE5](https://registry.igem.org/parts/bba-25vceoe5)). You may refer to our [Design | Description](https://2025.igem.wiki/hkust/description#design) page for further detail. Our principle design was based upon maximising the function of PflQ2MT for cadmium adsorption. As the cell tolerance to cadmium for both *E. coli* and *P. putida* were not well-characterised, nor was the rate or mechanism which Cd2+ entered the cell, our team believed a surface-expression of PflQ2MT using the Lpp-OmpA system would provide greater potential for protein binding and cell stability.

To drive high and controlled protein synthesis, we selected the T7 promoter. This promoter system is widely utilized in *E. coli* expression strains (which carry the T7 RNA Polymerase) due to its ability to achieve high expression levels rapidly, simplifying the overall circuit design and leveraging readily available, popular plasmid backbones. For predictable translation initiation, the Ribosome Binding Site (RBS) was chosen from the well-documented Elowitz library, ensuring reliable and robust protein production. Furthermore, to enhance the functionality and modularity of the fusion protein, we inserted two critical sequences between the Lpp-OmpA anchor and the PflQ2MT cargo. The first is the TEV (Tobacco Etch Virus) protease cleavage site. This site was selected after screening several candidates because it serves a dual purpose: it is essential for verifying successful surface expression through protease digestion assays, and it introduces modularity, allowing the concept to be easily extended to display binding domains for other heavy metals.

![Figure 4](https://static.igem.wiki/teams/5546/engineering/fig4.avif)

*Figure 4.* The screening process of protease cleavage site. From general meeting on 9th May.

To ensure maximum accessibility and efficiency of the TEV protease, a flexible (Glycine)6 linker (GGGGGG) was strategically inserted between the anchor protein and the cleavage site. This linker provides spatial separation and conformational flexibility, which significantly enhances the cutting efficiency of the TEV protease (Chen et al., 2016). For the mean of simplest, we also select T7 terminator.

Plasmids play a significant role in successful genetic engineering. Specifically, we selected pET11a plasmid to express the main circuit optimized for *E. coli*. pET11a demonstrates high-level expression of recombinant proteins in *E. coli*, utilizing the strong T7 promoter system. As a widely used plasmid, it contains Ampicillin resistance, MCS, and pBR322 ori. We used it to assemble our Main Circuit ([BBa\_25OWO9RF](https://registry.igem.org/parts/bba-25owo9rf)) into plasmid ([BBa\_25JXOJIK](https://registry.igem.org/parts/bba-25jxojik)) optimized for effective expression in *E. coli* DH5a and Rosetta.

![Figure 5](https://static.igem.wiki/teams/5546/engineering/fig3.avif)

*Figure 5.* pET-11a ([BBa\_25OZD053](https://registry.igem.org/parts/bba-25ozd053)). Standard plasmid in *E. coli* T7 expression system (not compatible with *P. putida*), ampicillin resistance, pBR322 ori, high copy number, MCS, Donated to us by our advisor Changxin, Expresses Main–*E. coli*-Optimized–Circuit.

You may like to take a look at our Cycle 1 Circuit Design logbook below:

[Cycle 1 Circuit Design Logbook](https://static.igem.wiki/teams/5546/engineering/cdlog/cd-cycle1.pdf)

Build

Our work involved assembling our composite part in the vector pET11a and expressing the plasmid in *E. coli* BL21. We also aimed to characterise its expression and downstream function in *E. coli*. Our goals for this cycle included confirming its expression and membrane localisation as well as its function as a metal-binding protein. For more details about the protocol design, please refer to the [Experiments](https://2025.igem.wiki/hkust/experiments) and [Protocols](https://2025.igem.wiki/hkust/protocols) page.

In this cycle, we also aimed to verify the correct folding, stability and binding under our desired conditions using Dry Lab. This would involve modelling the Lpp-OmpA-MT circuit into biologically accurate *E. coli* membranes and using GROMACs to generate trajectory files for further analysis. To see more details on this, please refer to the “MT Cycle 1” tab under our [Model](https://2025.igem.wiki/hkust/model) page.

Test

With the addition of His-tag downstream our MT, we detected the expression of MT through Western Blotting with monoclonal and HRP-conjugated secondary antibodies. From the experiment, bands of 23 kDa showed that PflQ2MT was successfully expressed in *E. coli*. Further, we tested whether the presence of MT increases the survivability of the bacteria compared to the wild type by the growth curve of *E. coli* (EC) and *E. coli* with Metallothionein (ECMT). Moreover, a seven-amino acid-long sequence (Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser) recognized specifically by TEV protease, was inserted between Lpp-OmpA ([BBa\_K1694010](https://registry.igem.org/parts/bba-k1694010)) and MT sequences and to verify the surface expression of the proteins by successful cleavage. In addition, Inductively-Coupled Plasma Optical Emission Spectrometry (ICP-OES) was utilized to test out the functionality of the MTs by measuring the concentrations of cadmium before and after ECMT treatment. Also, a survival test between wild-type *E. coli* and *E. coli* with our MT construct transformed. The details of the protocols and experiments are recorded in the [Experiments](https://2025.igem.wiki/hkust/experiments) page.

Learn

Higher final OD600 values from the growth curve suggest that *E. coli* with the metal-binding protein shows better survivability in the cadmium environment. However, both *E. coli* and *E. coli* with metallothionein did not show growth as reported in papers, while we suspected that it was due to their inability to adapt in an environment of heavy metals.

Generally readily available *E. coli* strains, like C+, K12, DH5ɑ, and Rosetta(DE3), were obtained from our lab stock. Eventually, we leveraged two common strains, including *E. coli* DH5ɑ for efficient plasmid transformation and cloning, and *E. coli* Rosetta(DE3) for high-level protein expression, particularly due to its compatibility with the T7 expression system, according to Du et al. (2021). The use of these standard strains allowed for rapid prototyping and verification of key genetic components before transitioning to a chassis better suited for the final application environment.

After more research, we noticed *P. putida* KT2440 as a better candidate as our chassis which exhibited more ideal characteristics and was more well-researched (Martínez-García & De Lorenzo, 2023). This decision was driven by several critical advantages that align with the project's real-world application in heavy metal remediation of industrial sludge. *P. putida* KT2440 offers superior environmental relevance, as it is a soil bacterium naturally found in environments similar to our target sludge (De Lorenzo et al., 2024). Furthermore, comparative studies demonstrated that *P. putida* exhibits significantly better resistance to the high-cadmium concentrations and acidic conditions characteristic of the target environment than *E. coli* (Higham et al., 1985). Experimentally, *P. putida* also showed a superior growth rate and higher viability at the relatively lower incubation temperature of 30 ℃, and demonstrated greater overall resistance to metabolic stress. These combined factors made *P. putida* the optimal host for developing our system.

## Cycle 2

---

For this cycle, we need to transform our chassis from *E. coli* to *P. putida*. Within such a transformation, we need to redesign the nearly whole workflow, including our circuit design, wet lab protocols, and more, while remaining our fundamental concept unchanged, to better improve our Main Circuit for a better fit of practical use, and to verify our concept on our new chassis.

Cycle 2: Metallothionein in *P. putida*
Expand All

## Cycle 2.1

*P. putida* KT2440 is a bacterium found in sludge. Due to its natural habitat, it has resistance towards high concentrations of heavy metals and extreme pH levels. *P. putida* showed characteristics of a chassis that was capable of fulfilling our goals of extracting cadmium within sludge.

Design

To further ensure that *P. putida* was a suitable chassis, we planned another cell viability assay to compare its survival compared to *E. coli.* Within sludge, there exists a vast array of heavy metal ions, contaminants, and slightly acidic conditions (pH 4-5). Thus, this required a more robust chassis that was resistant to such conditions. Also, we determined the optimal *P. putida* growth environment with 30℃, as well as constructed a *P. putida* transformation protocol, documented in our [Protocols](https://2025.igem.wiki/hkust/protocols).

Build

Our new chassis *P. putida* KT2440 was kindly provided by Prof. Conghui You from ShenZhen University, whose group is working on catabolic pathways in *Pseudomonas putida*. In order to ensure the expression of protein could occur in sludge, our goal was to determine whether the bacteria would survive in the concentration range where Cd2+ was found in sludge. Furthermore, we aimed to characterise the extent of resistance *P. putida* had in sludge. For further details, please refer to the [Experiments](https://2025.igem.wiki/hkust/experiments) page.

Test

Similarly to before, we prepared different concentrations of Cd2+ solution and allowed *P. putida* and *E. coli* to grow. Their absorbances were read periodically every 30 minutes. Their end OD600 absorbances were recorded and compared analytically. Particularly, *P. putida* was able to survive significantly better at 30 micromoles concentration of Cd2+ and persisted well up to 60 micromoles concentration of Cd2+. For more insights on the results, please refer to the [Experiments](https://2025.igem.wiki/hkust/experiments) page.

Learn

From the assay, we observed that *P. putida* indeed had a better survivability in cadmium compared to *E. coli*. From the previous literature review, Cd2+ concentration in sludge was found to be around 4 mg/L. Thus, this showed that *P. putida* was able to endure the desirable range and was thus a more suitable circuit to express the MT.

## Cycle 2.2

After confirming *P. putida* has better resistance than in our practical environment, we started to transform our Main Circuit, which was already verified in *E. coli*, to our new selected chassis. This transformation includes new parts, new vectors, new protocols, and a new mindset.

Design

In this cycle, our work was to transform the MT into *P. putida.* We integrated our MT main circuit into pSEVA441 and transformed the whole plasmid into *P. putida*. Compared to *E. coli*, *P. putida* KT2440 is not a chassis as popular in synthetic biology, and the T7 expression system does not function. We obtained the RBS, promoter, and terminator libraries of *P. putida* KT2440 from different articles (Elmore et al., 2017; Benedetti et al., 2021; Silva-Rocha & de Lorenzo, 2023) and utilized compatible Standard European Vector Architecture (SEVA) vectors for protein expression in *P. putida* KT2440 from previous studies. Additionally, rather than an IPTG-induced promoter, we also aimed to test whether *P. putida* could survive the metabolic burden if the MT was placed downstream to a constitutive promoter. Under such circumstances, we selected EM7 promoter ([BBa\_K4278705](https://registry.igem.org/parts/bba-k4278705)), pJL1-sfGFP RBS ([BBa\_25TMR0E8](https://registry.igem.org/parts/bba-25tmr0e8)), and eT1 terminator ([BBa\_25RQDKM3](https://registry.igem.org/parts/bba-25rqdkm3)) to construct our circuit ([BBa\_250SPQMJ](https://registry.igem.org/parts/bba-250spqmj)).

pSEVA441 is a standard cloning plasmid with spectinomycin resistance, MCS, and pRO1600 ori. It functions in both *E. coli* (DH5a and Rosetta (DE3)) and *P. putida* KT2440

We used pSEVA441 to express the Main Circuit optimized for *P. putida* KT2440.

![Figure 6](https://static.igem.wiki/teams/5546/engineering/fig4.avif)

*Figure 6.* pSEVA441 ([BBa\_2543S1TA](https://registry.igem.org/parts/bba-2543s1ta)). Cloning Plasmid Works in both *E. coli* and *P. putida*, spectinomycin resistance, MCS, pRO1600 ori, low copy number, expresses Main–*P. putida* KT2440-Optimized–Circuit. Gratefully obtained from SEVA.

You may like to take a look at our Cycle 2 Circuit Design logbook below:

[Cycle 2 Circuit Design Logbook](https://static.igem.wiki/teams/5546/engineering/cdlog/cd-cycle2.pdf)

Build

As a slight alteration to Cycle 1, we transformed the circuit in the vector pSEVA441 which appeared to show greater native compatibility to *P. putida*. Following Gibson Assembly, transformation into *E. coli* DH5a was first done, and the miniprep product was extracted. Finally, the plasmid was transformed into *P. putida* using electroporation. For more details, please refer to the [Experiments](https://2025.igem.wiki/hkust/experiments) page.

Test

Similarly to before, we prepared different concentrations and left *P. putida* and *E. coli* growing in them and read their absorbances every 30 minutes. Their end OD600 absorbances were recorded and compared analytically. Western Blot was done and confirmed that the circuit was able to constitutively express in *P. putida*. TEV protease cleavage assay was performed, with confirmed capability of cleavage site. Regrettably, time restrictions resulted in an inability to definitively confirm its surface expression in *P. putida*, nor was a metal-binding assay using ICP-OES done, which will be implemented in our future plans. For more insights and results, please refer to the [Experiments](https://2025.igem.wiki/hkust/experiments) page.

Learn

Our results with *P. putida* showed it provided more robust characteristics while still being able to successfully express the MT. Thus, our work with *E. coli* shows promising potential that the MT could carry out the same functionality in *P. putida*. With future plans, our team hopes our results show future potential for a cadmium-specific bioremediation solution. Also, the enhanced capabilities of *P. putida* allowed us to not only transfer the Main Circuit but also to design and integrate two additional, application-critical circuits: a dedicated cadmium Biosensor and a Kill Switch for safety.

## Cycle 3

---

After transitioning to a more optimal chassis *P. putida* and successfully retrieving our Main Circuit for cadmium surface binding, we took a deeper look into how to fit into the need of a practical use as a one-stop solution towards cadmium remediation from sludge. Eventually, we came up with an extraordinary idea, which is to add a cadmium biosensor into our chassis, for the indication of the remediation progress.

Cycle 3: Biosensor
Expand All

## Cycle 3.1

To construct a biosensor, we followed a fundamental logic, starting from an input, then processing, eventually transferring to a readable output. Within this logic, our Biosensor circuit has gone through multiple times of iteration for optimal design and part selection.

Design

In our project, we aimed to develop a biosensor capable of monitoring cadmium concentrations in real time. Existing cadmium biosensors typically face two major limitations:

1. They respond slowly to changes in metal concentration.
2. Their signals increase with higher metal levels but cannot decrease once activated.

This led to our ideation of creating a biosensor which reacts in real-time. Achieving a genuine real-time response requires optimizing the kinetics of the fluorescent reporter protein in two critical ways: fast expression (to rapidly generate a large signal) and rapid degradation (to ensure quick signal clearance, preventing carry-over from previous measurements and allowing for accurate, dynamic monitoring).

We initially focused on the degradation aspect by incorporating a degradation tag at the C-terminus of our reporter protein. Literature reviews, which highlighted the effectiveness of tags like the LVA degradation tag confirmed this strategy. Implementing a faster degrading reporter system is essential for making our biosensor suitable for real-world, continuous monitoring applications.

Concurrently, to control and fine-tune the expression level, we utilized the Ptrc-lacO promoter. This IPTG-inducible system allows us to precisely adjust the expression rate, balancing the need for high output with the metabolic load on the cell. Furthermore, for the reporter component itself, we introduced a new, improved variant of the fluorescent protein mScarlet-I3-NCwt into the iGEM Registry ([BBa\_25IVGUN2](https://registry.igem.org/parts/bba-25ivgun2)). Characterization and testing of this improved reporter are vital steps in validating the performance of our real-time biosensor system.

For details of the biosensor design, please refer to our [Design | Description](https://2025.igem.wiki/hkust/description#design) page.

pSEVA234 with the lacI-Ptrc system was chosen as an expression vector for *P. putida* KT2440. pSEVA234 has Kanamycin resistance, MCS, and pBBR1 ori, demonstrating high expression efficiency in both *P. putida* KT2440 and *E. coli*. We used pSEVA234 to express the biosensor circuit and the kill-switch circuit.

![Figure 7](https://static.igem.wiki/teams/5546/engineering/fig5.avif)

*Figure 7.* pSEVA234 ([BBa\_25XDR5JO](https://registry.igem.org/parts/bba-25xdr5jo)). Expression plasmid, lacI-Ptrc system, works in both *E. coli* and *P. putida*, kanamycin resistance, MCS, pBBR1 ori, medium-high copy number, expresses the Biosensor Circuit and the Kill Switch. Gratefully obtained from SEVA.

You may like to take a look at our Cycle 3.1 Circuit Design logbook below:

[Cycle 3.1 Circuit Design Logbook](https://static.igem.wiki/teams/5546/engineering/cdlog/cd-cycle3-1.pdf)

Build

To make sure that our biosensor design actually works, we first designed a testing circuit for the mScarlet and the LVA degradation tag. The circuit is regulated by IPTG, allowing us to alter the expression level in order to test fluorescence and degradation at ease.

![Figure 8](https://static.igem.wiki/teams/5546/engineering/fig6.avif)

*Figure 8.* Demonstration of our Biosensor Degradation Test Circuit.

Test

The main focus of the first Biosensor cycle is to test the parts that are reviewed in the literature but not yet tested extensively. In the preliminary testing, we tested where the LVA tag added to the mScarlet works with IPTG induction. After the induction, IPTG was washed away with PBS to test out the degradation rate of fluorescence. Please refer to our [Experiment](https://2025.igem.wiki/hkust/experiments) page and the ‘[Biosensor Degradation Test](https://static.igem.wiki/teams/5546/protocols/biosensor-degradation-test-protocol.pdf)’ protocol.

Learn

From the results of the degradation test, we were confident that the fluorescent protein (mScarlet) worked as expected. Though the degradation of the fluorescent signal was not obvious after washing away IPTG, we believed that the main reason might be the leakage of the promoter (since the fluorescence signal was detected even before adding IPTG). This urges us to look for other effective degradation systems. Despite such flaws in our report system, we still went forward, to design a Biosensor with other properties facilitating real-time detection.

## Cycle 3.2

After a few iterations, we ultimately discovered and selected the Toehold Switch system as the processing system, and the cadR system as our input. We successfully constructed our full Biosensor, and made efforts in its testing and characterization. With a well-documented future plan on testing the sensitivity of the regulatory system, our Biosensor became a powerful tool for our genetic modified machine to be applied practically.

Design

We went on to further test the whole biosensing system. In addition to the LVA tag, the [RiboSwitch](https://registry.igem.org/collections/27076eb8-1906-4e4e-ac8e-c62fa958ea88) was designed from Toehold Switch, which will lead to mRNA encoding the mScarlet continuously being transcribed in the absence of cadmium. With the upstream regulatory and input system of tetR ([BBa\_259S6D4J](https://registry.igem.org/parts/bba-259s6d4j), regulated by anhydrotetracycline (aTc)) and cadR ([BBa\_251WLXJ9](https://registry.igem.org/parts/bba-251wlxj9)), we successfully composed our full Biosensor circuits ([BBa\_256ORVBQ](https://registry.igem.org/parts/bba-256orvbq)).

![Figure 9](https://static.igem.wiki/teams/5546/engineering/fig7.avif)

*Figure 9.* Demonstration of our full Biosensor circuit, excelling in real-time capability and sensitivity.

[Cycle 3.2 Circuit Design Logbook](https://static.igem.wiki/teams/5546/engineering/cdlog/cd-cycle3-2.pdf)

Build

Our full Biosensor circuit was built with three fragments, and divided into two inserts. With the pSEVA234 Gibson Assembly overhangs ([BBa\_25I4ZGKJ](https://registry.igem.org/parts/bba-25i4zgkj), [BBa\_2506U5S6](https://registry.igem.org/parts/bba-2506u5s6)), in addition to the insert overhang i ([BBa\_2534D5XF](https://registry.igem.org/parts/bba-2534d5xf)), the Biosensor was assembled into pSEVA234 and our Biosensor plasmid ([BBa\_25EHA43F](https://registry.igem.org/parts/bba-25eha43f)) was built.

Test

From the preliminary test, we detected the red mScarlet fluorescence, when the *P. putida* with a full Biosensor circuit (PPfBS) was exposed to 50 µM cadmium solutions. *E. coli* with the same circuit (ECfBS) was transformed successfully and incubated with the same concentration of cadmium, but it did not glow. Furthermore, we measured the fluorescence of PPfBS incubated in 50 µM cadmium solutions overnight by FlexStation. You may check our results and interpretations via our [Experiment](https://2025.igem.wiki/hkust/experiments) page.

Learn

We suspect that the reason for not detecting fluorescence from ECfBS was due to growth conditions and circuit design, as both bacteria were incubated at 30°C and circuit codons are optimized for *P. putida*, which are not ideal for *E. coli* to show reproducible results. We plan to repeat the experiment with lower cadmium concentrations to test out the regulatory system of aTc and the sensitivity of the whole circuit.

We still could not detect the degradation of the fluorescence from the overnight measured data, which suggests the LVA degradation tag was not working well. A wired but characterized result was obtained and documented in our [Experiment](https://2025.igem.wiki/hkust/experiments) page, which indicates the fluorescence intensity decreases then increases with cadmium concentration rising. We also inferred that a complicated construct might have burdened the bacteria, which led us to design the circuit into a more simplified version.

## Cycle 4

---

In order to implement our genetic engineered machine into practical use, a mechanism to ensure safety and trigger cell death after leaking into the environment is vital. Upon this concept, we designed a Kill Switch to ensure biosafety for our chassis.

Cycle 4: Kill Switch
Expand All

Design

Besides building the biosensor, we also designed a kill switch to prevent the persistence of genetically modified bacteria in case of leakage. For details, please refer to our [Design | Description](https://2025.igem.wiki/hkust/description#design) page.

During our attendance of iGBA, a regional iGEM conference, a senior member of BNUZH-China Team, iGEM 2024, introduced to us an inducible promoter PopdH ([BBa\_K5291021](https://registry.igem.org/parts/bba-k5291021)) and its corresponding expression system needed to adjust the expression of proteins for our Kill Switch circuit. When targeting the kill system,, we selected lysis protein E ([BBa\_K2152003](https://registry.igem.org/parts/bba-k2152003)) originating from bacteriophage Phi X 174, to maximise lysis effectiveness and ensure no leakage.

In the beginning ,we weren’t entirely sure whether PodpH is compatible with our chassis *P. putida* due to limited literature, therefore we compared the structure of key proteins involved in the regulation of PodpH. Through protein structure predictions and protein alignment, we determined that PodpH is cross-compatible. See “Kill Switch Modelling” in our [Model](https://2025.igem.wiki/hkust/model) page.

![Figure 10](https://static.igem.wiki/teams/5546/engineering/fig8.avif)

*Figure 10.* Demonstration of our Kill Switch, preventing the persistence of the genetic engineered machine after leaking into natural environment.

Build

Our Kill Switch circuit was built with four fragments, and divided into three inserts. With the pSEVA234 Gibson Assembly overhangs ([BBa\_25I4ZGKJ](https://registry.igem.org/parts/bba-25i4zgkj), [BBa\_2506U5S6](https://registry.igem.org/parts/bba-2506u5s6)), in addition to the insert overhang i ([BBa\_2534D5XF](https://registry.igem.org/parts/bba-2534d5xf)) and the unique site ([BBa\_25ZHOKVG](https://registry.igem.org/parts/bba-25zhokvg)), we could selectively adding the lysis gene in and assemble the whole device ([BBa\_25CTXIKQ](https://registry.igem.org/parts/bba-25ctxikq)) into pSEVA234 and construct our Kill Switch plasmid ([BBa\_258P32V0](https://registry.igem.org/parts/bba-258p32v0)).

Test

Initially, 1 mM IPTG and 2 mM citric acid were used to test the Kill Switch construct, as suggested by the literature. We plated the transformed bacteria on an agar plate with Kanamycin, which led to no growth of bacteria. Then, we switched the method of testing, making IPTG and citrate-containing agar plates instead of adding them to the liquid bacterial culture. This showed a promising result, where *E. coli* with the Kill Switch construct grew.

Learn

With the promising result, we will test out with different concentrations of IPTG and citric acid to see the expression levels and characterize the circuit. Our preliminary result shows that the Kill Switch works logically as expected, while still pending to implement a detailed characterization to determine if it aligns well with the safety needs of the practical application.

## Future Cycle

---

Due to time constraints within the initial project cycle, several key validation steps and advanced design implementations remain slated for future work. Our continued efforts will focus on fully characterizing our latest constructs and translating our engineered mechanism into a practical, real-world application pipeline.

Future Cycle
Expand All

Design

The next phase of design focuses on rigorous characterization and modular improvement:

#### **1. Real-World Characterization**

Driven by insights from our Human Practices efforts, the most critical next step is comprehensive characterization. This includes assessing the efficiency of our biosorbent when applied to real sludge samples and validating the proposed application within a functional pipeline, details of which are further elaborated on our [Project Description](https://2025.igem.wiki/hkust/description#application) page.

#### **2. Improved Cadmium Binding Constructs**

Following consultation with Prof. BAE and based on dry lab screening results, we designed an improved version of our cadmium surface binding circuit ([BBa\_25310AKD](https://registry.igem.org/parts/bba-25310akd)). This design replaces the previously used PflQ2MT with EC20, a phytochelatin we were initially unable to synthesize. You may like to take a look at our EC20 Circuit Design logbook below:

[EC20 Circuit Design Logbook](https://static.igem.wiki/teams/5546/engineering/cdlog/cd-ec20.pdf)

Subsequent to the successful implementation and characterization of EC20, we plan to further enhance the cadmium binding capacity and specificity by integrating a specifically designed peptide sequence (Li et al., 2025).

#### **3. Modularity and Extensibility**

A core feature of our design is its modularity, allowing the basic concept to be extended to remediate other heavy metals:

* **Extension of Concept:** By simply changing the heavy metal binding protein in the main circuit, our system can be adapted. For instance, based on our interview with Biometallica, targeting arsenic, which a major heavy metal pollutant affecting rice crops—is a high-priority extension.
* **Heavy Metal Sorting:** By incorporating different heavy metal binding proteins alongside corresponding, specific protease cleavage sites (each recognized by a different cleavage factor), we could potentially realize a system capable of heavy metal sorting.
* **Biosensor Adaptation:** Our biosensor circuit can also be adapted to detect various other heavy metals by changing the metal-dependent promoter and ion-responsive element.

Build

In the current cycle, we successfully built the following constructs intended for future testing:

* **Improved Cadmium Binding Circuit (**[BBa\_254H79TD](https://registry.igem.org/parts/bba-254h79td)**):** We successfully assembled the fragments for the improved circuit featuring the EC20 binding domain.

![Figure 11](https://static.igem.wiki/teams/5546/engineering/fig9.avif)

*Figure 11.* Demonstration of EC20 cadmium surface binding design, which shows promising capability for a more specific and effective binding.

* **Simplified Cadmium Biosensor:** A simplified version of our cadmium biosensor circuit was also constructed.

![Figure 12](https://static.igem.wiki/teams/5546/engineering/fig10.avif)

*Figure 12.* Demonstration of simplified Biosensor design, showing similar reacting behavior, despite losing part of the real-time ability and quick response.

Test

The next phase is dedicated to rigorous testing and moving toward real-world application:

#### **1. EC20 Characterization**

While we successfully assembled the fragments for the EC20 circuit ([BBa\_25310AKD](https://registry.igem.org/parts/bba-25310akd)), we unfortunately did not have sufficient time to conduct the necessary expression experiments and binding characterization. Thorough testing of this improved construct is a priority.

#### **2. Column Pipeline Validation**

We have a detailed plan to validate the proposed column pipeline through small-scale experiments and characterization studies. This will involve:

* Characterizing the efficiency of the biosorbent within the flow system.
* Estimating the operational cost.
* Optimizing parameters such as flow rate and regeneration cycles.

#### **3. Real-World Implementation**

The ultimate goal is to implement our technology in a real-life setting. This will involve utilizing real sludge samples to characterize the system's efficiency under complex environmental conditions. We aim to collaborate with end-users, such as the local facility T-Park, to conduct large-scale trials. This collaboration will be crucial for fine-tuning our pipeline and processing protocol to specifically target the composition of local sludge.

## References

---

+ Calero, P., Jensen, S. I., & Nielsen, A. T. (2016). Broad-Host-Range ProUSER Vectors Enable Fast Characterization of Inducible Promoters and Optimization of p-Coumaric Acid Production in Pseudomonas putida KT2440. *ACS Synthetic Biology*, *5*(7), 741–753. <https://doi.org/10.1021/acssynbio.6b00081>
+ Chen, Y., Li, Q., Yang, J., & Xie, H. (2016). Promoting TAG Removal of a MBP-Fused Integral Membrane protein by TEV protease. *Applied Biochemistry and Biotechnology*, *181*(3), 939–947. <https://doi.org/10.1007/s12010-016-2260-z>
+ De Lorenzo, V., Pérez-Pantoja, D., & Nikel, P. I. (2024). Pseudomonas putida KT2440: the long journey of a soil-dweller to become a synthetic biology chassis. *Journal of Bacteriology*, *206*(7). <https://doi.org/10.1128/jb.00136-24>
+ De Siqueira, G. M. V., & Guazzaroni, M. (2023). Host-Dependent Improvement of GFP Expression in Pseudomonas putida KT2440 Using Terminators of Metagenomic Origin. *ACS Synthetic Biology*, *12*(5), 1562–1566. <https://doi.org/10.1021/acssynbio.3c00098>
+ Du, F., Liu, Y., Xu, Y., Li, Z., Wang, Y., Zhang, Z., & Sun, X. (2021). Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production. *Microbial Cell Factories*, *20*(1). <https://doi.org/10.1186/s12934-021-01680-6>
+ Higham, D. P., Sadler, P. J., & Scawen, M. D. (1985). Cadmium Resistance in Pseudomonas putida: Growth and Uptake of Cadmium. *Microbiology*, *131*(10), 2539–2544. <https://doi.org/10.1099/00221287-131-10-2539>
+ *INP-NC*. (n.d.). [https://parts.igem.org/Part:BBa\\_K3279006](https://parts.igem.org/Part%3ABBa%5C_K3279006)
+ Krogsgaard, M., Wucherpfennig, K. W., Canella, B., Hansen, B. E., Svejgaard, A., Pyrdol, J., Ditzel, H., Raine, C., Engberg, J., & Fugger, L. (2000). Visualization of myelin basic protein (MBP) T cell epitopes in multiple sclerosis lesions using a monoclonal antibody specific for the human histocompatibility leukocyte antigen (HLA)-DR2–MBP 85–99 complex. *The Journal of Experimental Medicine*, *191*(8), 1395–1412. <https://doi.org/10.1084/jem.191.8.1395>
+ Li, J., Fan, C., Lau, H. Y., & Wu, T. (2025). A modular fusion neural network approach to efficiently predict Multi-Metal binding sites in protein sequences. *bioRxiv (Cold Spring Harbor Laboratory)*. <https://doi.org/10.1101/2025.09.13.676010>
+ *Lpp-OmpA*. (n.d.). [https://parts.igem.org/Part:BBa\\_K1694002](https://parts.igem.org/Part%3ABBa%5C_K1694002)
+ Martínez-García, E., & De Lorenzo, V. (2023). Pseudomonas putida as a synthetic biology chassis and a metabolic engineering platform. *Current Opinion in Biotechnology*, *85*, 103025. <https://doi.org/10.1016/j.copbio.2023.103025>
+ Martin-Pascual, M., Batianis, C., Bruinsma, L., Asin-Garcia, E., Garcia-Morales, L., Weusthuis, R. A., Van Kranenburg, R., & Santos, V. a. M. D. (2021). A navigation guide of synthetic biology tools for Pseudomonas putida. *Biotechnology Advances*, *49*, 107732. <https://doi.org/10.1016/j.biotechadv.2021.107732>
+ Narita, J., Okano, K., Tateno, T., Tanino, T., Sewaki, T., Sung, M., Fukuda, H., & Kondo, A. (2005). Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion. *Applied Microbiology and Biotechnology*, *70*(5), 564–572. <https://doi.org/10.1007/s00253-005-0111-x>
+ Parks, L., Ek, M., Ståhl, S., & Löfblom, J. (2024). Investigation of an AIDA-I based expression system for display of various affinity proteins on Escherichia coli. *Biochemical and Biophysical Research Communications*, *696*, 149534. <https://doi.org/10.1016/j.bbrc.2024.149534>
+ Tas, H., Goñi-Moreno, Á., & De Lorenzo, V. (2020). A standardized inverter package borne by broad host range plasmids for genetic circuit design in Gram-Negative bacteria. *ACS Synthetic Biology*, *10*(1), 213–217. <https://doi.org/10.1021/acssynbio.0c00529>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

* [Cell viability assay](#cell-viability)
* [Western Blot](#western-blot)
* [Protein surface expression - TEV Protease cleavage](#protein-surface)
* [Cadmium binding assay - ICP-OES](#cadmium-binding)
* [Biosensor and Kill Switch Results](#experiment-result)

![Background Image](https://static.igem.wiki/teams/5546/title/3.webp)

### The survivability of *P. putida* is better than *E. coli* in a high concentration of Cd2+ proved by a cell viability assay

---

#### **Aim**

The aim of this experiment is to assess and compare the survivability of  *E. coli*  and  *P. putida*  with or without expressing metallothionein (MT) under different concentrations of cadmium. The strains we used include wild-type  *E. coli*  BL21(EC),  *E. coli*  BL21 expressing metallothionein (ECMT), wild-type  *P. putida*  KT2440 (PP), and  *P. putida*  KT2440 expressing MT (PPMT). This experiment proved that  *P. putida*  KT2440 survived better than  *E. coli*  BL21 in a high concentration of cadmium (> 30 µM), which is chosen as our chassis. Meanwhile, the expression of MT increases the survivability of both bacteria.

#### **Experimental design**

Bacteria preparation:

* Three colonies of each sample were picked and inoculated in 5 mL of LB without antibiotics for 16 hours at 37°C.
* The overnight culture is subcultured at a 1:50 ratio into 50 mL LB at 37°C until OD600 reaches ~1.0.
* The expression of MT in  *E. coli*  was induced by IPTG during subculturing for 4 hours.
* The subcultures are diluted 100 times before cadmium is added.
* OD600 was measured after incubating for 9 hours.

Cadmium dilution:

* A serial dilution was done on the cadmium stock solution using LB as the diluent.
* 0 µM to 480 µM was used as the range of final concentration of cadmium solution, as referenced from a study (Kim et al., 2020).

Plate reading:

* OD600 was measured using a Flexstation 3 Multi-Mode Microplate Reader.
* Kinetic mode was used to obtain data points at a 20-minute interval for 19 hours and 20 minutes.

#### **Results**

The average OD600 was calculated from triplicates, and a heat map was plotted with the concentration of cadmium solution against the OD600 of different types of bacteria.

![Figure 1.](https://static.igem.wiki/teams/5546/experiments/experimentfig1.webp)

**Figure 1.** Heat map of cadmium concentration against OD600 of different types of bacteria.

![Figure 2.](https://static.igem.wiki/teams/5546/experiments/od600-for-30um-cd.webp)

**Figure 2.** Bar chart of OD600 of EC and PP at 30 µM Cd2+. Student’s t-test ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3.

![Figure 3.](https://static.igem.wiki/teams/5546/experiments/experimentsfig3.webp)

**Figure 3.** Bar chart of OD600 of different types of bacteria at 240 µM Cd2+. Student’s t-test ∗P < 0.05, n = 3.

From the gradient in colour between EC and ECMT (30 µM to 240 µM), it can be observed that ECMT has a higher OD600 than EC, which justifies that MT does increase the survival of bacteria in cadmium solutions (Figure 1, 3). What’s more, at 30 µM Cd2+,  *P. putida*  has a significantly larger OD600 than  *E. coli*  (Figure 2).

Hence, it is concluded that the metallothionein protein increases the survivability of the bacteria, and  *P. putida*  is a more advantageous candidate as a chassis to remove cadmium. However, we still need to prove the successful expression of MT in both  *E. coli*  and  *P. putida*  (see next experiment).

#### **Future Plan**

Different parameters, such as different pH levels, citrate concentrations, and IPTG induction time, can be applied in the experiment design and further characterize the survivability of each bacterial strain.

### The successful expression of MT in *E. coli* and *P. putida* verified by Western Blot

---

#### **Aim**

The aim of this experiment is to confirm the expression of metallothionein (MT) in  *E. coli*  BL21 and  *P. putida*  KT2440.

#### **Experimental design**

MT is constructed with a His-tag, so anti-His-tag mouse monoclonal antibody (sc-8036, Santa Cruz) is used as the primary antibody, and goat anti-mouse
IgG-Horseradish Peroxidase (HRP) (sc-2005, Santa Cruz) is used as the secondary antibody.

Protein Sample Preparation:

* Both  *E. coli*  and  *P. putida*  were inoculated overnight for approximately 14-16 hours (*E. coli*  was grown at 37°C while  *P. putida*  at 30°C).
* The expression of MT in  *E. coli*  was induced with IPTG for 4 hours.
* Bacteria were centrifuged, and the cell pellet was lysed with RIPA lysis buffer.
* Supernatant was collected, and cell pellets were resuspended in the remaining supernatant in tubes.
* 6X SDS Loading buffer was added to both supernatant and cell pellet resuspension, and the samples were boiled.

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE):

* A polyacrylamide gel was set up and run (4% stacking and 12% separating gels).

Western Blot:

* Anti-His-tag mouse monoclonal primary antibody.
* Goat anti-mouse IgG-HRP secondary antibody.

#### **Results**

![Figure 4.](https://static.igem.wiki/teams/5546/experiments/experiment-wb-ecmt-result.webp)

**Figure 4.** Western blot result of  *E. coli*  with metallothionein (ECMT) induced by different concentrations of IPTG. Ponceau S is used as the loading control.

![Figure 5.](https://static.igem.wiki/teams/5546/experiments/screenshot-2025-10-07-at-12-31-39-am.webp)

**Figure 5.** Western blot result of  *P. putida*  with metallothionein (PPMT).
An expected band of 23 kDa was shown.

From Figure 4, it was observed that the production of metallothioneins increased with increasing amounts of IPTG, as indicated by the stronger detected signals. It also proved the successful expression of MT in  *E. coli* . In addition, MT was also detected in  *P. putida*  after electroporation of the plasmid (Figure 5). We successfully expressed the fusion proteins with our circuits in both chassis –  *E. coli*  and  *P. putida.* However, the MT was shown in the cell precipitation. Based on our design, Lpp-OmpA-MT should be expressed on the cell membrane. One explanation is that MT is attached to some membrane debris and is not soluble. Next, we need to prove the membrane localization of MT (see next experiment).

#### **Future Plan**

We can quantify the Western blot bands with standard curves to get the protein expression level.

### Lpp-OmpA-MT is expressed on the bacterial cell membrane verified by TEV protease cleavage

---

#### **Aim**

The aim of this experiment is to verify the expression of the fusion proteins on the bacterial surface and ensure they are in the correct orientation. This experiment is done with TEV protease and performing Western Blot to detect the cleaved peptides.

#### **Experimental design**

Bacteria preparation:

* 1 colony of  *E. coli*  BL21 with metallothionein (ECMT) was picked and inoculated in 5 mL of LB without antibiotics at 37°C overnight.
* The next day, the OD600 absorbance of the overnight culture was checked with an absorbance reader.
* Based on the absorbance, the overnight culture was diluted to an OD600 of ~0.5-0.6.
* The diluted overnight culture was then subcultured with 5 mL of fresh LB with antibiotics and IPTG to induce protein expression.
* The subculture was incubated for 1 to 2 hours at 37°C for more growth.
* The subculture was then centrifuged to harvest the cell pellet.
* The cell pellet was washed twice with 50 mM Tris-HCl, pH 7.5.
* The cell pellet was then resuspended in the same buffer, and TEV protease was added at a ratio of 1 unit per OD unit.
* The cells were incubated overnight at 4°C with slow rotation on a belly dancer.

Sample preparation:

* The cells were centrifuged, and the supernatant and pellet were collected.
* SDS-PAGE and Western blot were performed to see the cleaved metallothionein proteins.

#### **Expected Results**

Cleaved MT of ~7 KDa will be detected in the supernatant.

#### **Results**

![Figure 6.](https://static.igem.wiki/teams/5546/experiments/experimentfig6.webp)

**Figure 6.** Western blot result of ECMT TEV cleavage test

![Figure 7.](https://static.igem.wiki/teams/5546/experiments/experimentfig7.webp)

**Figure 7.** Western blot result of PPMT TEV cleavage test

After incubation of bacterial culture with TEV protease overnight at 4°C, supernatant and cell pellets were separated to detect the cleaved MT in the supernatant as expected. However, we could not detect the cleaved MT with a size of 7 kDa in the supernatant but found the whole MT of 23 kDa, one band at around 17 kDa, and one band at the bottom (Figure 6). To note, the separating gel of SDS-PAGE was 12%, where the range of target proteins’ molecular weights is from 10 to 250 kDa. This suggests that the bands at the bottom can be the cleaved MT but not separated well by SDS-PAGE. Interestingly, the results show the presence of TEV protease at 37 kDa when the literature expected its size to be 27 kDa. Considering the reproducibility of the bands and the different composition of the SDS-PAGE gel, we concluded that the 37 kDa band is TEV protease. The results show TEV protease cleavage works, but the cleaved MTs and the enzymes are found in the sample precipitation, which suggests some interactions happened to pull down TEV protease and cleaved MTs. We suspect that some lipopolysaccharide (LPS) or lipoproteins may interact with them.

#### **Future Plan**

Based on the results to date, we observed that cleaved MTs are found in the precipitation after TEV protease cleavage. We suspect that the cleaved MTs interact with some LPS or proteins on the bacteria surface, because TEV protease is also in the precipitation. More repeats need to be conducted. Furthermore, the cell membrane can be separated through the membrane fractionation to strengthen our conclusion of the localization of fusion proteins. In addition, Tricine-SDS-PAGE will be used to separate the cleaved peptides or the percentage of the crosslinker will be increased.

### The functionality of MT is proved by cadmium binding assay - ICP-OES

---

#### **Aim**

The aim of this experiment is to confirm the function of the metallothionein expressed in  *E. coli*  BL21, to the metal, cadmium, by using Inductively-Coupled Plasma Optical Emission Spectrometry (ICP-OES).

#### **Experimental design**

Bacteria preparation:

* *E. coli*  BL21 expressing metallothionein (ECMT) and wild-type  *E. coli*  BL21 (ECWT) were picked and inoculated in 5 mL of LB with the appropriate antibiotic selection for 16 hours at 37°C and 250 RPM shaking incubator.
* Due to past empirical results showing reduced growth (overnight OD ~0.8-0.9), each inoculum consisted of 2-3 colonies to increase the growth rate.

Sample preparation:

* OD600 of the overnight culture was measured. Readings reached ~1.0-1.5.
* The overnight culture was subcultured into three groups: ECMT with IPTG (20mL), ECMT without IPTG (20mL), and ECWT (20mL).

+ The overnight culture was subcultured at a 1:20 ratio into 20 mL of LB at 37°C until OD600 reached ~0.5-0.7.
+ Each group had 3 subcultures for triplicate. A total of 9 cultures were prepared.
+ 2 mM of IPTG was added for induction (ECMT with IPTG) and incubated for 4-5 hours at 37°C in a 250 RPM Shaking Incubator.

* After incubation, each subculture was separated into 4 Falcon tubes with 5 mL each. (Total of 9 tubes).
* The subculture was washed twice with 1x PBS. Each wash was done at 3000 x g for 10 - 15 minutes.
* The cell pellets were resuspended and stored in 1 mL 1x PBS solution overnight due to working hour restrictions.
* The next day, cells were centrifuged at 3000 x g for 15 minutes to re-pellet. Supernatant was removed.
* 1 mg/mL Cadmium Solution was first diluted in 2-5% Nitric Acid to produce a 240 µM Cd2+ solution. From there, dilutions of 0, 20, 40, 60, 80, 100, and 120 µM Cd2+ Solution.
* Cell pellets were resuspended in 3-4 mL of 60 µM Cd2+ solution and incubated at 37°C in a shaking incubator at 230 RPM.
* Half the samples (3 tubes of each group) were immediately centrifuged at 15000 x g for 15 minutes to sample for t0 readings.
* Samples were also then filtered through a 0.22 micron PES Filter.

tO/N samples were prepared in parallel by allowing the incubation in 60 µM Cd2+ solution to occur overnight.

#### **Expected Results**

There will be a decrease in the concentration of Cd2+ solution in the samples after incubation.

#### **Results**

![Figure 8.](https://static.igem.wiki/teams/5546/experiments/ecmt-icp-oes-result-prism.webp)

**Figure 8.** ICP-OES result of  *E. coli*  with different treatments in a 60 µM Cd2+ solution. n = 2-3.

After the concentration of Cd2+ was detected by ICP-OES, we normalized the data with OD600 to see the difference in the concentrations of the three bacterial samples.  *E. coli*  with MT shows a higher decrease in cadmium concentration, which proposes its potential to bind Cd2+ and extract it from the solutions.

#### **Future Plan**

We observed a dramatic decrease of cadmium concentrations with ECMT. However, the variation among samples caused the result not to be significant (Figure 8). Therefore, we will repeat the experiment to obtain more data to make it consistent and significant. The experiment will also be conducted in  *P. putida*  with the identical sample preparation and experiment design to verify the functionality of the metallothioneins in  *P. putida'* s main circuit. A higher decrease of cadmium concentrations is expected compared to  *E. coli,*  because  *P. putida*  has a better survivability in high Cd2+ solutions (Figure 1).

### Biosensor and Kill Switch Results

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Experimental data for Biosensor and Kill Switch can be found below.

[Biosensor & Kill Switch Experimental Data](https://static.igem.wiki/teams/5546/experiments/biosensor-and-kill-switch-results-1.pdf)

### References

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+ Hollinger, J., Wu, J., Awayda, K. M., O'Connell, M. R., & Yao, P. (2023). Expression and purification of the mitochondrial transmembrane protein FAM210A in *Escherichia coli*. Protein expression and purification, 210, 106322. <https://doi.org/10.1016/j.pep.2023.106322>
+ How to enhance detection limits in icp-oes for trace metal analysis—Drawell. (2024, December 27). <https://www.drawellanalytical.com/how-to-enhance-detection-limits-in-icp-oes-for-trace-metal-analysis/>
+ ICP-OES sample preparation—Us. (n.d.)., from <https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/spectroscopy-elemental-isotope-analysis-learning-center/trace-elemental-analysis-tea-information/icp-oes-information/icp-oes-sample-preparation.html>
+ Kim, A., Park, S., & Sung, J. H. (2020). Cell Viability and Immune Response to Low Concentrations of Nickel and Cadmium: An In Vitro Model. International Journal of Environmental Research and Public Health, 17(24), 9218. <https://doi.org/10.3390/ijerph17249218>
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