Abstract

Heavy metals in water pose serious health risks. Our team engineered E. coli to express rice metallothionein (OsMTI-1b) for metal binding and a urease gene cluster from Sporosarcina pasteurii for Microbiologically Induced Calcite Precipitation (MICP) to remove heavy metal ions from water. In tests, both OsMT1 and the urease gene cluster functioned effectively in our engineered bacteria, significantly increasing the removal rates of Cd²⁺ and Pb²⁺. The strain expressing both genes, OsMT1-Ure, showed the best overall performance, with OsMT1 being more effective at lower concentrations and the urease gene cluster being more efficient at higher concentrations, achieving a maximum removal rate of 85.78% for cadmium and 98.98% for lead. Our engineered DH5α strain is scalable, eco-friendly, and ideal for use in industrial wastewater treatment plants.

DBTL Cycle 1:

1. Design

1.1 Introduction: Heavy metals

Heavy metals, such as lead, mercury, cadmium, and chromium, are extensively released into the environment through industrial activities, mining, and waste disposal. They pose serious health risks to humans, including neurotoxicity, nephrotoxicity, and carcinogenicity, while also harming ecosystems and disrupting biodiversity (Abd Elnabi et al., 2023; Balali-Mood et al., 2021; Tchounwou et al., 2012). In our hometown, the Yangtze River Delta of China, studies identified significant ecological risks from mercury, cadmium, and chromium, with carcinogenic risk indices up to 24,800 times higher than safe levels (Mei-juan et al., 2018; Qinxuan et al., 2018; Zhan-jun et al., 2018). Reports indicate that other provinces in China, particularly Guangxi, Fujian, and Liaoning, suffer from more severe cadmium and lead pollution than the Yangtze River Delta (Wu et al., 2022; Yu et al., 2017). Chemical precipitation is the most commonly used method for heavy metal removal. However, we believe bioremediation is the future due to its sustainability and cost-effectiveness. As an iGEM team, we aim to engineer bacteria to enhance its bioremediation efficiency.

1.2 The chassis: Escherichia coli DH5α & BL21(DE3)

E. coli is a safe and well-developed platform that is suitable for expressing exogenous genes. We chose E. coli for its ease of use and potential for further development. Acknowledging that our project may not be perfect, we hope future teams can build upon our work easily.

Specifically, we used E. coli DH5α and BL21(DE3).

DH5α is the most commonly used E. coli strain for molecular cloning and plasmid amplification. It is a highly efficient competent cell strain and is known for its enhanced DNA stability. However, its protein expression levels are relatively low compared to other E. coli strains.

BL21(DE3) is a strain designed for high-level protein expression, especially with plasmids using a T7 promoter. Lack of proteases like OmpT and Lon reduces protein degradation, making it ideal for producing large quantities of intact proteins (Jeong et al., 2015).

1.3 The Metallothionein: OsMTI-1b

Metallothioneins (MTs) are a class of small, cysteine-rich proteins found in a wide range of organisms, including mammals, plants, and microorganisms (Coyle et al., 2002). The primary function of them is to bind and sequester metal ions within cells. MTs achieve metal binding through the presence of an abundance of cysteine residues, which possess a high affinity for metal ions, allowing MTs to tightly bind a variety of metals (Carpene et al., 2007; Klaassen et al., 1999). As a result, MTs are involved in various physiological processes, including metal ion transport, antioxidant defense, maintaining metal ion concentrations at appropriate levels, and most importantly, absorbing metal ions to prevent metal-induced toxicity (Ruttkay-Nedecky et al., 2013; Si & Lang, 2018; Thirumoorthy et al., 2007).

Figure 1. The general structure of metallothionein. Four and three atoms of metal ions are coordinated in α- and β-domains, respectively (Klaassen et al., 1999).

Introducing MTs into our engineered cells is expected to have double advantages in terms of heavy metal removal. Firstly, MTs can bind to heavy metal ions in solution and lower their concentrations directly (He et al., 2024; Lu et al., 2023). Secondly, since heavy metal ions are also toxic to bacteria and can hinder their growth or even kill them, MTs play a pivotal role in enhancing the tolerance of cells to heavy metal ions, enabling the engineered bacteria to function in concentrated polluted water.

Rice (Oryza sativa), like many other organisms, has metallothioneins (MTs) for metal ion homeostasis and detoxification. Due to rice’s status as a model organism and a staple crop, its metallothioneins (OsMTs) are well-studied and are famous for their high efficiency of absorbing heavy metal ions (Cobbett & Goldsbrough, 2002). Eleven protein isoforms of metallothioneins are found in rice (Fang et al., 2010; Gautam et al., 2012). In previous studies, OsMTI-1b (noted as OsMT1 in this project) has shown the best metal ion absorption abilities among them (Ansarypour & Shahpiri, 2017; Shahpiri & Mohammadzadeh, 2018).

Figure 2. Map of Non-OmpA-OsMT1.

OsMTI-1b is a relatively small protein by itself, consisting of only 72 amino acids (Buell et al., 2005). In order to maximize its expression level and metal-binding effect, we planned to express it on the surface of our engineered bacteria by fusing a signal-like sequence (Non) and the extracellular part of the E. coli outer membrane protein A (OmpA, amino acid 46-159) (Earhart, 2000) to the N-terminus of OsMTI-1b, making the fusion protein 20.67 kDa (Figure 3). The Non-OmpA system, developed by Jeiranikhameneh et al. in 2017, is a surface expression system utilizing the non-classical secretion pathway. It offers higher efficiency compared to the traditional Lpp-OmpA system.

1.4 The enzymes: Urease gene cluster from Sporosarcina pasteurii

Microbiologically Induced Calcite Precipitation (MICP) is a biomineralization process driven by microorganisms, primarily bacteria, that catalyze the formation of calcium carbonate minerals. This phenomenon often occurs through the metabolic activity of ureolytic bacteria, which possess urease enzymes (Bachmeier et al., 2002; Sarayu et al., 2014). In normal MICP, urease catalyzes the hydrolysis of urea, a common organic compound found in many environments, into ammonia and carbonate ions. The ammonia generated by this reaction raises the pH of the surrounding environment, while the carbonate ions react with calcium ions (or other cations) present in the solution to form insoluble calcium carbonate precipitates. These calcite precipitates accumulate around the bacterial cells and on nearby surfaces, gradually forming solid mineral deposits (Wu et al., 2021).

Figure 3. A. Schematic of microbially induced calcite precipitation (MICP) catalyzed by S. pasteurii cells. (Wu et al., 2021); B. Map of the urease gene cluster of S. pasteurii DSM33 (Pei et al., 2023).

We believe MICP holds promise for heavy metal removal from water because the precipitation process can happen with almost all metal ions. For example, when cadmium ions instead of calcium ions are present in the environment, cadmium carbonate will be produced as a precipitate, removing the cadmium ions from the solution and lowering the toxicity (Qasem et al., 2021; Zeng et al., 2021). To conclude, MICP is a versatile method capable of removing a wide range of heavy metals, which is perfect for our project.

Sporosarcina pasteurii, formerly known as Bacillus pasteurii, is a Gram-positive bacterium renowned for its ability to induce biomineralization processes, being one of the strongest ureolytic bacteria in nature (Wu et al., 2021; Yoon et al., 2001). S. pasteurii is commonly found in soil and other natural environments, where it plays a significant role in ecological processes and microbial communities due to its ability to change the microenvironment. Moreover, S. pasteurii is safe for humans, animals, and plants, so we believe it can be a great gene donor for our project (Omoregie et al., 2019).

The strain DSM 33 is the type strain of S. pasteurii whose whole genome was sequenced and analyzed (GenBank accession: GCA_041295575.1). As a result, we were able to locate its urease genes, which are responsible for MICP, design primers, and amplify them. The good news for us is that its urease genes are located very closely, forming a gene cluster so we can acquire all the genes together by a single PCR. The urease gene cluster of S. pasteurii DSM 33 is around 5300 bp long, consisting of seven genes, named ureA, ureB, ureC, ureE, ureF, ureG, and ureD (Pei et al., 2023; You et al., 1995).

UreA, ureB, and ureC encode the gamma, beta, and alpha structural subunits of the urease enzyme complex (Moersdorf et al., 1994). UreC, the alpha subunit, is the key catalytic component responsible for urea hydrolysis. It contains the active site and exhibits the highest expression level among the subunits (You et al., 1995). The urease is a nickel-containing enzyme. UreE, ureF, and ureD encode nickel-binding proteins that are essential for the assembly of the urease metallocentre (Ciurli et al., 2002; Remaut et al., 2001). Additionally, ureG encodes a GTPase that hydrolyzes GTP to provide the energy required for the insertion of nickel ions into the metallocentre (Zambelli et al., 2005). All the genes would be introduced into E. coli.

1.5 Plasmid selection: Modified pET-28a

For exogenous gene expression in E. coli, we found the pET-28a vector to be very effective (Figure 4). It contains a high-copy origin of replication, enabling strong gene expression and easy plasmid extraction. Additionally, the kanamycin resistance marker is advantageous in experiments, as kanamycin is more stable and reliable than ampicillin, another commonly used selection antibiotic. While the T7 promoter is highly potent and makes pET-28a ideal for gene expression in general laboratory settings, it requires inducers to function. This is unsuitable for our project, as our proposed application is in water treatment plants where inducers are impractical.

Figure 4. Plasmid map of pET-28a(+).

To address this, we selected a strong constitutive promoter, BBa_J23100, from the iGEM Registry. Known as the strongest in its family, its effectiveness has been validated by multiple iGEM teams (Anderson, 2006). Its short length (35 bp) and broad compatibility (compatible with RCF[10], [21], [23], [25], and [1000]) are additional advantages. We planned to incorporate BBa_J23100 into the pET-28a vector to promote the expression of OsMTI-1b and the urease gene cluster. Additionally, we planned to insert a strong RBS, BBa_J61101, from the iGEM Registry upstream of the target genes (Anderson, 2007).

2. Build

2.1 Recombinant vector construction

The BBa_J23100 promoter, BBa_J61101 RBS, the Non-OmpA cell membrane expression system, and the codon-optimized OsMT1 gene were chemically synthesized in a row by GENEWIZ (Suzhou, Jiangsu, China) and inserted between the XbaI and BamHI restriction sites. The resulting recombinant vector was pET28a-OsMT1 (Figure 6A).

 

Figure 5. Gel electrophoresis of PCR product of the urease gene cluster.

The 5338 bp urease gene cluster was amplified by PCR (Phusion High-Fidelity PCR Master Mix, Thermo Fisher, Waltham, MA, USA) using 1 µL of S. pasteurii DSM33 liquid culture as the template (Figure 5). The initial denaturation step was extended to 10 minutes to ensure complete cell lysis and release of the genomic DNA template. pET28a-OsMT1 was then digested with NheI and XhoI (FastDigest, Thermo Fisher, Waltham, MA, USA) to remove OsMT1. Gibson Assembly (ClonExpress Ultra One Step Cloning Kit, Vazyme, Nanjing, Jiangsu, China) was performed to insert the urease gene cluster between the restriction sites, resulting in pET28a-Ure (Figure 6B).

Figure 6. A. Gel electrophoresis of PCR products of part pET28a-Ure and part pET28a-OsMT1; B. Plasmid map of pET28a-OsMT1; C. Plasmid map of ET28a-Ure; D. Plasmid map of pET28a-OsMT1-Ure.

Finally, to assemble the complete plasmid, pET28a-OsMT1-Ure, both the partial pET28a-OsMT1 (4277 bp) and partial pET28a-Ure (6956 bp) were linearized by PCR. The partial pET28a-OsMT1 contains the OsMT1 fusion gene along with half of the plasmid backbone, including the origin of replication, promoter, and RBS. The partial pET28a-Ure contains the urease gene cluster and the other half of the plasmid backbone, including the T7 terminator and kanamycin resistance gene. On our first try, we managed to get the linearized partial pET28a-OsMT1 (4277 bp) but failed to get bands from partial pET28a-Ure (6956 bp), probably due to reduced PCR efficiency when amplifying a long fragment. We tried to adjust the template amount but it did not help. After several failed PCRs, we tried touchdown PCR from 65°C to 50°C, and the PCR products showed clear bands (Figure 6A). The two fragments were combined using Gibson Assembly to generate the complete pET28a plasmid containing both the OsMT1 and the urease gene cluster.

The recombinant vectors pET28a-OsMT1, pET28a-Ure, and pET28a-OsMT1-Ure were transformed into E. coli DH5α. Colony PCR was used to confirm successful vector construction, followed by sequencing verification (Figure 7). The verified plasmids were then extracted from DH5α and transformed into BL21(DE3), with colony PCR performed again to verify successful transformation.

Figure 7. Gel electrophoresis of colony PCR products for verification of successful vector construction and transformation in DH5α. Lanes 1-5: pET28a-OsMT1; Lanes 6-10: pET28a-Ure; Lanes 11-15: pET28a-OsMT1-Ure.

3. Test

3.1 Functional validation

For validation of gene expression and function, the easiest way is to test whether the strains induce precipitate formation. We prepared LB media containing CaCl₂ and urea and inoculated the engineered strains to culture overnight at 37 ℃.

Figure 8. Overnight culture of engineered E. coli in LB-urea-calcium media. A. E. coli BL21 harboring pET28a-Ure; B. E. coli DH5α harboring pET28a-Ure. The dark grains were calcium carbonate precipitates.

Results showed that only E. coli DH5α containing pET28a-Ure and pET28a-OsMT1-Ure facilitated calcium carbonate precipitation (Figure 8B). Interestingly, the E. coli BL21(DE3) containing the same vectors did not form carbonate precipitates (Figure 8A). This result is unexpected, as we anticipated higher gene expression in BL21 compared to DH5α, which should result in greater urease activity and consequently more precipitate formation in BL21.

3.2 SDS-PAGE analysis of cell lysates

To investigate what might have caused the gene to be non-functional in BL21, we decided to run SDS-PAGE with cell lysates. To be able to see the heat-sensitive fusion OsMT1 (membrane protein) in SDS-PAGE, we avoided boiling the samples. Instead, we opted for glass bead lysis on ice and incubation at 37°C.

Figure 9. SDS-PAGE of total protein of engineered E. coli DH5α (A) and BL21 (B).

Our SDS-PAGE results showed that although the engineered DH5α strains were functional, no significant changes were observed when running total cell protein on the gel (Figure 9A). In contrast, in BL21, we observed the presence of the introduced proteins, including ureC (the main subunit of the urease gene cluster) and the fusion protein OsMT1, confirming the high expression levels of these proteins (Figure 9B).

4. Learn

The absence of visibly detectable protein in E. coli DH5α is likely due to its naturally low protein expression levels of DH5α. This result is consistent with prior studies that confirmed the high efficiency of the urease enzyme complex (Sarayu et al., 2014; Wu et al., 2021), as its functionality was demonstrated despite the lack of visible protein bands in total protein SDS-PAGE.

 

Despite high expression levels, no ureolytic activity was observed in BL21. This may be due to the excessive expression rate of proteins in BL21. High expression likely overwhelmed the cell’s folding machinery, resulting in misfolded proteins or inclusion bodies, causing the proteins non-functional (Bhatwa et al., 2021; Rosano & Ceccarelli, 2014). This issue is especially prominent with large proteins like ureC (61.44 kDa) (Baneyx & Mujacic, 2004). Conversely, DH5α’s lower expression levels may have allowed the proteins more time to fold correctly, preserving their functionality.

DBTL Cycle 2:

1. Design

As discussed, the loss of function in E. coli BL21 was attributed to an excessively high expression rate. Although this issue could potentially be mitigated by adjusting culture conditions, such as lowering the temperature (Bhatwa et al., 2021; Sørensen & Mortensen, 2005), we decided to give up BL21-based strains because we believe in stability. In real-world applications, we do not want our bacteria to lose function simply because they are under high-temperature conditions. Additionally, DH5α is superior in plasmid stability, which is advantageous for practical use.

Therefore, we plan to only use DH5α-based strains in future tests.

2. Build

The construction of pET28a-OsMT1, pET28a-Ure, and pET28a-OsMT1-Ure in E. coli DH5α has been completed in the first cycle.

3. Test

3.1  Growth Curve

First, we aimed to ensure that our engineered E. coli DH5α could still grow normally and that the introduced proteins were not toxic to the cells. Each strain was inoculated into flasks containing LB media and cultured at 37°C. OD600 measurements were taken at 0, 2, 4, 6, 8, 24, 28, and 32 hours.

Figure 10. Growth curves of E. coli DH5α-based strains in liquid LB for 32h.

The results showed that all strains had similar growth rates, indicating that the expression of OsMT1 and the urease gene cluster did not significantly affect cell metabolism. This confirmed the scalability and potential for real-world application of our engineered strains.

3.2 Hg Removal

Next, we conducted tests on actual heavy metal removal, starting with Hg²⁺. We prepared LB media containing mercury(II) nitrate concentrations ranging from 0 to 10 mM in 10-fold dilutions. A 1% inoculum of the engineered E. coli was subcultured into each mercury-containing medium and incubated overnight at 37 °C for 24 hours. On day 2, OD600 measurements were taken for each sample (Figure 11A).

The results showed that at 0.0001 mM Hg²⁺, the OD600 was similar to the control without mercury, while higher Hg²⁺ concentrations resulted in no growth for all strains. This indicates that the minimum inhibitory concentration (MIC) of Hg²⁺ for our strains lies between 0.0001 mM and 0.001 mM. No increase in mercury tolerance was observed in the engineered strains compared to the original DH5α. However, this may not be conclusive, as we only tested 10-fold dilutions, meaning any resistance increase smaller than 10-fold might have been missed. In fact, there are reports that expressing exogenous metallothioneins enhances heavy metal tolerance in E. coli (Qinxuan et al., 2018; Xu et al., 2018). Therefore, in future experiments, testing a narrower range of concentrations will be necessary to detect any small increases in heavy metal tolerance. Additionally, the maximum tolerance concentration determines the applicable concentration range of our engineered bacteria in real-world conditions. For Hg²⁺ removal, the applicable concentration range is 0 to 0.0001 mM.

Figure 11. A. Growth of E. coli DH5α-based strains in liquid LB containing Hg²⁺; B. Removal rates of Hg²⁺ by E. coli in 24 hours. “N/A” stands for “not applicable,” as no data was collected from 0.001 to 10 mM due to the absence of cell growth.

The supernatants from the 24-hour cultures were collected and sent to Convinced-test Tech. Co., Ltd (Nanjing, Jiangsu, China) for Hg²⁺ concentration analysis. Mercury removal rates were calculated and are shown in Figure 11B.

The results indicated that at a very low concentration of 0.0001 mM, all strains demonstrated a similar mercury removal rate of about 87%, suggesting that the expression of OsMT1 and the urease gene cluster did not enhance mercury removal at the concentration the bacteria could tolerate. These findings align with previous reports that E. coli can also absorb mercury and other metal ions (Bae et al., 2001; Shahpiri & Mohammadzadeh, 2018). The results confirmed that even unmodified DH5α can effectively remove a significant percentage of mercury ions at concentrations as low as 0.0001 mM, indicating that the intrinsic mercury binding/absorption systems were not saturated at such low concentration levels. As a result, although the engineered strains may possess enhanced heavy metal binding and precipitation abilities, this effect was not evident in the Hg²⁺ tests at 0.0001 mM, and higher mercury concentrations could not be tested due to the inhibition of cell growth. Further testing will explore the impact of OsMT1 and the urease gene cluster on cadmium and lead removal.

3.3 Cd Removal

Cadmium removal tests were conducted following the same protocol as for mercury. After 24 hours, OD600 was measured for each tube (Figure 13A). The results showed that all strains were able to grow in Cd²⁺ concentrations ranging from 0.0001 to 0.1 mM. No growth was observed at 1 mM of Cd²⁺, indicating that the MIC for Cd²⁺ in our strains lies between 0.1 mM and 1 mM. 0.1 mM represents the upper limit of the applicable concentration range for real-world applications. As discussed, while tolerance to Cd²⁺ may have increased, it was not detectable in our 10-fold dilution tests. Interestingly, at a very high concentration of 10 mM, CdCl₂ inhibited bacterial growth and spontaneously formed a large amount of flocculent precipitate, as shown in the right tube in Figure 12. We suspect that these precipitates are Cd(OH)Cl and Cd(OH)₂ (Bazarkina et al., 2010).

Figure 12. Flocculent precipitates formed in the tubes on the right containing 10 mM CdCl₂.

Figure 13. A. Growth of E. coli DH5α-based strains in liquid LB containing Cd²⁺; B. Removal rates of Cd²⁺ by E. coli in 24 hours. “N/A” stands for “not applicable,” as no data was collected from 1 to 10 mM due to the absence of cell growth.

The Cd²⁺ concentration in the supernatant of each tube was measured, and the removal rates are shown in Figure 13B.

The results indicated that at a very low concentration of 0.0001 mM cadmium, all strains demonstrated a similar removal rate of approximately 80%, suggesting that the intrinsic metal-binding/absorbing systems were functioning effectively and had not yet reached saturation. For the original DH5α, the intrinsic metal-binding/absorbing systems became saturated at 0.001 mM cadmium, and minimal absorption was observed at higher concentrations. In OsMT1, the cadmium binding capacity increased significantly. At 0.001 mM, it exhibited the highest removal rate of 88.53% and maintained a high removal rate of 74.60% at 0.1 mM. However, the binding capacity of the OsMT1 protein on the cell surfaces was eventually saturated at 0.1 mM. In summary, OsMT1 was most effective at cadmium concentrations below 0.01 mM. For Ure, there was no noticeable change in removal rate compared to DH5α at 0.001 mM, likely due to the low efficiency of the chemical reaction (formation of cadmium carbonate) when the concentration of one reactant is very low ("Rate of reaction," 1997). The removal rate increased as the cadmium concentration rose from 0.01 to 0.1 mM, reaching 55.04%. In conclusion, urease is more suitable for high-concentration conditions and was most effective when cadmium concentrations exceeded 0.1 mM. Lastly, for OsMT1-Ure, it appears that both mechanisms acted independently, as the removal rates were close to the sum of the individual performances of OsMT1 and Ure.

3.4 Pb Removal

Lead removal tests were conducted using the same protocol as for mercury and cadmium. OD600 results confirmed that all strains were able to grow in Pb²⁺ concentrations ranging from 0.0001 to 1 mM, but no growth was observed at 10 mM, indicating that the MIC for Pb²⁺ in these strains lies between 1 mM and 10 mM (Figure 14A). This suggests that 1 mM of Pb²⁺ is the upper limit for practical applications. Similar to CdCl₂, 10 mM Pb(NO₃)₂ formed significant flocculent precipitate, suspected to be PbCl₂ and Pb(OH)₂ (Lide, 2004; Yoshida et al., 2003).

 

Figure 14. A. Growth of E. coli DH5α-based strains in liquid LB containing Pb²⁺; B. Removal rates of Pb²⁺ by E. coli in 24 hours. “N/A” stands for “not applicable,” as no data was collected on 10 mM due to the absence of cell growth.

The Pb²⁺ removal rates were similar to Cd²⁺. At a low concentration of 0.0001 mM, all strains showed a similar removal rate of around 90%, driven by the intrinsic metal-binding/absorbing systems. This intrinsic removal rate was higher than that observed for Hg²⁺ and Cd²⁺, suggesting that E. coli has a higher affinity for lead. In DH5α, saturation occurred at 0.001 mM, with minimal absorption at higher concentrations. In OsMT1, removal rates exceeded 95% at 0.001 and 0.01 mM, but saturation occurred at 0.1 mM. Ure exhibited no significant difference from DH5α at low concentrations but showed a high increase in removal rate at higher concentrations (0.1 to 1 mM), peaking at 96.25%. OsMT1-Ure showed the best overall performance, with OsMT1 handling lower concentrations and Ure managing higher concentrations, making the combination more effective across a wider range of lead concentrations.

3.5 Removal Rate

Based on previous tests, we found that OsMT1-Ure was the most effective strain for heavy metal removal, efficiently managing both low and high concentrations. In this experiment, we tested the removal rate by time, by adding Cd²⁺ and Pb²⁺ to overnight cultures of E. coli DH5α expressing OsMT1-Ure. Samples were taken every 2 hours to monitor the changes in the removal rate.

Figure 15. Changes in the removal rate of Cd²⁺ and Pb²⁺ by E. coli DH5α expressing OsMT1-Ure.

Results showed that at a low concentration of 0.01 mM, the removal of both lead and cadmium was completed in less than 2 hours. At this concentration, the removal was primarily due to OsMT1 binding to the heavy metal ions directly, which explains the rapid reaction. At a higher concentration of 0.1 mM, where the urease gene cluster was dominating, the removal took about 4 hours due to the time required for urea hydrolysis. These findings provided valuable information about reaction time, which guided the design of our device, a reaction and sedimentation tank for use in wastewater treatment plants. This experiment also reconfirmed that E. coli has a higher bioaffinity for lead than cadmium.

4. Learn

The introduction of OsMT1 and the urease gene cluster into E. coli DH5α did not affect normal cell growth. Both OsMT1 and the urease gene cluster functioned effectively in our engineered bacteria, dramatically increasing the removal rates of Cd²⁺and Pb²⁺ in the culture. The strain expressing both genes, OsMT1-Ure, exhibited the best overall performance, with OsMT1 being more effective at lower concentrations and the urease gene cluster being more efficient at higher concentrations, making this combination more effective across a wider range of heavy metal levels. The upper concentration limits for heavy metal removal were 0.0001 mM for Hg²⁺, 0.1 mM for Cd²⁺, and 1 mM for Pb²⁺.

5. Future plans

As mentioned in the Test section, the engineered strains showed no increase in heavy metal tolerance compared to the original DH5α. However, this result may not be definitive since we only tested 10-fold dilutions, potentially missing smaller changes in resistance. Future experiments should use a finer concentration range to detect subtle tolerance increases. This should also determine the precise applicable concentration range.

Our second plan is to enhance biosafety for the engineered E. coli. It is essential to prevent the accidental release of GMOs into the environment. Given that the activity of the urease gene cluster in our engineered bacteria relies on the presence of urea, we plan to implement a suicide mechanism that turns on when urea is absent. The suicide switch can be achieved by utilizing UreR, a family of transcriptional regulators from ureolytic bacteria, which is activated in the presence of urea (Dattelbaum et al., 2003). By placing a repressor gene under its control, the repressor is expressed in the presence of urea, preventing cell suicide. In the absence of urea, the repression is lifted, triggering the suicide mechanism and causing the bacteria to self-destruct. This design should ensure the bacteria can only survive within the wastewater treatment plant tanks.

References