iGEM 2024 — Team hangzhou-sdg

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{% block title %}Notebook{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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Engineering Success

**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
andBL21(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).

![](https://static.igem.wiki/teams/5205/engineering/0926/1.png)

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).

![](https://static.igem.wiki/teams/5205/engineering/0926/2.png)

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).

![](https://static.igem.wiki/teams/5205/engineering/0926/3.png)

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.

![](https://static.igem.wiki/teams/5205/engineering/0926/4.png)

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).

![](https://static.igem.wiki/teams/5205/engineering/0926/5.png)

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).

![](https://static.igem.wiki/teams/5205/engineering/0926/6.png)

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.

![](https://static.igem.wiki/teams/5205/engineering/0926/7.png)

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 ℃.

![](https://static.igem.wiki/teams/5205/engineering/0926/8.jpg)

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.

![](https://static.igem.wiki/teams/5205/engineering/0926/9.jpg)

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.

![](https://static.igem.wiki/teams/5205/engineering/0926/10.png)

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.

![](https://static.igem.wiki/teams/5205/engineering/0926/11.png)

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).

![](https://static.igem.wiki/teams/5205/engineering/0926/12.jpg)

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

![](https://static.igem.wiki/teams/5205/engineering/0926/13.png)

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).

![](https://static.igem.wiki/teams/5205/engineering/0926/14.png)

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.

![](https://static.igem.wiki/teams/5205/engineering/0926/15.png)

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**

Abd Elnabi, M. K., Elkaliny, N. E., Elyazied, M. M., Azab, S. H.,
Elkhalifa, S. A., Elmasry, S., Mouhamed, M. S., Shalamesh, E. M.,
Alhorieny, N. A., Abd Elaty, A. E., Elgendy, I. M., Etman, A. E.,
Saad, K. E., Tsigkou, K., Ali, S. S., Kornaros, M., & Mahmoud, Y.
A. (2023). Toxicity of Heavy Metals and Recent Advances in Their
Removal: A Review. *Toxics*,*11*(7). <https://doi.org/10.3390/toxics11070580>

Anderson, J. (2006). *Part:BBa\_J23100*. Retrieved June 17
from [https://parts.igem.org/Part:BBa\_J23100](https://parts.igem.org/Part%3ABBa_J23100)

Anderson, J. (2007). *Part:BBa\_J61101*. Retrieved
September 10 from [https://parts.igem.org/Part:BBa\_J61101](https://parts.igem.org/Part%3ABBa_J61101)

Ansarypour, Z., & Shahpiri, A. (2017). Heterologous expression of
a rice metallothionein isoform (OsMTI-1b) in Saccharomyces cerevisiae
enhances cadmium, hydrogen peroxide and ethanol tolerance. *Braz J Microbiol*,*48*(3), 537-543. <https://doi.org/10.1016/j.bjm.2016.10.024>

Bachmeier, K. L., Williams, A. E., Warmington, J. R., & Bang, S.
S. (2002). Urease activity in microbiologically-induced calcite
precipitation. *J Biotechnol*,*93*(2),
171-181. [https://doi.org/10.1016/s0168-1656(01)00393-5](https://doi.org/10.1016/s0168-1656%2801%2900393-5)

Bae, W., Mehra, R. K., Mulchandani, A., & Chen, W. (2001). Genetic
engineering of Escherichia coli for enhanced uptake and
bioaccumulation of mercury. *Appl Environ Microbiol*,*67*(11), 5335-5338. <https://doi.org/10.1128/aem.67.11.5335-5338.2001>

Balali-Mood, M., Naseri, K., Tahergorabi, Z., Khazdair, M. R., &
Sadeghi, M. (2021). Toxic Mechanisms of Five Heavy Metals: Mercury,
Lead, Chromium, Cadmium, and Arsenic. *Front Pharmacol*,*12*, 643972. <https://doi.org/10.3389/fphar.2021.643972>

Baneyx, F., & Mujacic, M. (2004). Recombinant protein folding and
misfolding in Escherichia coli. *Nat Biotechnol*,*22*(11), 1399-1408. <https://doi.org/10.1038/nbt1029>

Bazarkina, E. F., Pokrovski, G. S., Zotov, A. V., & Hazemann,
J.-L. (2010). Structure and stability of cadmium chloride complexes in
hydrothermal fluids. *Chemical Geology*,*276*(1), 1-17. [https://doi.org/https://doi.org/10.1016/j.chemgeo.2010.03.006](https://doi.org/https%3A/doi.org/10.1016/j.chemgeo.2010.03.006)

Bhatwa, A., Wang, W., Hassan, Y. I., Abraham, N., Li, X. Z., &
Zhou, T. (2021). Challenges Associated With the Formation of
Recombinant Protein Inclusion Bodies in Escherichia coli and
Strategies to Address Them for Industrial Applications. *Front Bioeng Biotechnol*,*9*, 630551. <https://doi.org/10.3389/fbioe.2021.630551>

Buell, C. R., Yuan, Q., Ouyang, S., Liu, J., Zhu, W., Wang, A., Maiti,
R., Haas, B., Wortman, J., Pertea, M., Jones, K. M., Kim, M., Overton,
L., Tsitrin, T., Fadrosh, D., Bera, J., Weaver, B., Jin, S., Johri,
S., . . . Jackson, S. (2005). Sequence, annotation, and analysis of
synteny between rice chromosome 3 and diverged grass species. *Genome Res*,*15*(9), 1284-1291. <https://doi.org/10.1101/gr.3869505>

Carpene, E., Andreani, G., & Isani, G. (2007). Metallothionein
functions and structural characteristics. *J Trace Elem Med Biol*,*21 Suppl 1*, 35-39. <https://doi.org/10.1016/j.jtemb.2007.09.011>

Ciurli, S., Safarov, N., Miletti, S., Dikiy, A., Christensen, S. K.,
Kornetzky, K., Bryant, D. A., Vandenberghe, I., Devreese, B., Samyn,
B., Remaut, H., & van Beeumen, J. (2002). Molecular
characterization of Bacillus pasteurii UreE, a metal-binding chaperone
for the assembly of the urease active site. *J Biol Inorg Chem*,*7*(6), 623-631. <https://doi.org/10.1007/s00775-002-0341-7>

Cobbett, C., & Goldsbrough, P. (2002). Phytochelatins and
metallothioneins: roles in heavy metal detoxification and
homeostasis. *Annu Rev Plant Biol*,*53*,
159-182. <https://doi.org/10.1146/annurev.arplant.53.100301.135154>

Coyle, P., Philcox, J. C., Carey, L. C., & Rofe, A. M. (2002).
Metallothionein: the multipurpose protein. *Cell Mol Life Sci*,*59*(4), 627-647. <https://doi.org/10.1007/s00018-002-8454-2>

Dattelbaum, J. D., Lockatell, C. V., Johnson, D. E., & Mobley, H.
L. (2003). UreR, the transcriptional activator of the Proteus
mirabilis urease gene cluster, is required for urease activity and
virulence in experimental urinary tract infections. *Infect Immun*,*71*(2), 1026-1030. <https://doi.org/10.1128/iai.71.2.1026-1030.2003>

Earhart, C. F. (2000). Use of an Lpp-OmpA fusion vehicle for bacterial
surface display. *Methods Enzymol*,*326*,
506-516. [https://doi.org/10.1016/s0076-6879(00)26072-2](https://doi.org/10.1016/s0076-6879%2800%2926072-2)

Fang, Y., Yang, H., Wang, T., Liu, B., Zhao, H., & Chen, M.
(2010). Metallothionein and superoxide dismutase responses to
sublethal cadmium exposure in the clam Mactra veneriformis. *Comp Biochem Physiol C Toxicol Pharmacol*,*151*(3), 325-333. <https://doi.org/10.1016/j.cbpc.2009.12.005>

Gautam, N., Verma, P. K., Verma, S., Tripathi, R. D., Trivedi, P. K.,
Adhikari, B., & Chakrabarty, D. (2012). Genome-wide identification
of rice class I metallothionein gene: tissue expression patterns and
induction in response to heavy metal stress. *Funct Integr Genomics*,*12*(4), 635-647. <https://doi.org/10.1007/s10142-012-0297-9>

He, N., Yao, W., & Tang, L. (2024). Surface expression of
metallothionein enhances bioremediation in Escherichia coli. *Desalination and Water Treatment*,*317*(6). <https://doi.org/10.1016/j.dwt.2024.100070>

Jeiranikhameneh, M., Razavi, M. R., Irani, S., Siadat, S. D., &
Oloomi, M. (2017). Designing novel construction for cell surface
display of protein E on Escherichia coli using non-classical pathway
based on Lpp-OmpA. *AMB Express*,*7*(1),
53. <https://doi.org/10.1186/s13568-017-0350-0>

Jeong, H., Kim, H. J., & Lee, S. J. (2015). Complete Genome
Sequence of Escherichia coli Strain BL21. *Genome Announc*,*3*(2). <https://doi.org/10.1128/genomeA.00134-15>

Klaassen, C. D., Liu, J., & Choudhuri, S. (1999). Metallothionein:
an intracellular protein to protect against cadmium toxicity. *Annual review of pharmacology and toxicology*,*39*, 267-294.

Lide, D. R. (2004). *CRC Handbook of Chemistry and Physics*.

Lu, C. W., Ho, H. C., Yao, C. L., Tseng, T. Y., Kao, C. M., &
Chen, S. C. (2023). Bioremediation potential of cadmium by recombinant
Escherichia coli surface expressing metallothionein MTT5 from
Tetrahymenathermophila. *Chemosphere*,*310*,
136850. <https://doi.org/10.1016/j.chemosphere.2022.136850>

Mei-juan, X., Bo, B., Chun-yan, C., Yong-chuan, W., Fu-yan, G., &
Xiao-yi, Y. (2018). Assessment of Heavy Metal Pollution and Human
Health Risk of Surface Waters in the City of Ningbo, China. *Environmental Science*,*39*(2), 729-737.

Moersdorf, G., Weinmann, P., & Kaltwasser, H. (1994). *Nucleotide sequence of three genes on a urease encoding
DNA-fragment from Bacillus pasteurii*.

Omoregie, A. I., Palombo, E. A., Ong, D. E. L., & Nissom, P. M.
(2019). Biocementation of sand by Sporosarcina pasteurii strain and
technical-grade cementation reagents through surface percolation
treatment method. *Construction and Building Materials*,*228*, 116828. [https://doi.org/https://doi.org/10.1016/j.conbuildmat.2019.116828](https://doi.org/https%3A/doi.org/10.1016/j.conbuildmat.2019.116828)

Pei, D., Liu, Z., & Hu, B. (2023). *A novel urease gene structure of Sporosarcina pasteurii with double
operons*.

Qasem, N. A. A., Mohammed, R. H., & Lawal, D. U. (2021). Removal
of heavy metal ions from wastewater: a comprehensive and critical
review. *npj Clean Water*,*4*(1), 36. <https://doi.org/10.1038/s41545-021-00127-0>

Qinxuan, S., Pengcheng, S., Weiping, F., Boyi, S., Dongren, Z., Jun,
W., Lei, X., & Qifang, W. (2018). Ecological risk and
bioaccumulation of heavy metals in the surface sediments in Qiantang
River. *Journal of shanghai ocean university*,*27*(5), 710-717. <https://doi.org/10.12024/jsou.20171102176>

Rate of reaction. (1997). In *IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold
Book") (1997). Online corrected version: (2006–)*. <https://doi.org/10.1351/goldbook.R05156>

Remaut, H., Safarov, N., Ciurli, S., & Van Beeumen, J. (2001).
Structural basis for Ni(2+) transport and assembly of the urease
active site by the metallochaperone UreE from Bacillus
pasteurii. *J Biol Chem*,*276*(52),
49365-49370. <https://doi.org/10.1074/jbc.M108304200>

Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein
expression in Escherichia coli: advances and challenges. *Front Microbiol*,*5*, 172. <https://doi.org/10.3389/fmicb.2014.00172>

Ruttkay-Nedecky, B., Nejdl, L., Gumulec, J., Zitka, O., Masarik, M.,
Eckschlager, T., Stiborova, M., Adam, V., & Kizek, R. (2013). The
role of metallothionein in oxidative stress. *Int J Mol Sci*,*14*(3), 6044-6066. <https://doi.org/10.3390/ijms14036044>

Sarayu, K., Iyer, N. R., & Murthy, A. R. (2014). Exploration on
the biotechnological aspect of the ureolytic bacteria for the
production of the cementitious materials--a review. *Appl Biochem Biotechnol*,*172*(5), 2308-2323. <https://doi.org/10.1007/s12010-013-0686-0>

Shahpiri, A., & Mohammadzadeh, A. (2018). Mercury removal by
engineered Escherichia coli cells expressing different rice
metallothionein isoforms. *Annals of Microbiology*,*68*(3), 145-152. <https://doi.org/10.1007/s13213-018-1326-2>

Si, M., & Lang, J. (2018). The roles of metallothioneins in
carcinogenesis. *J Hematol Oncol*,*11*(1),
107. <https://doi.org/10.1186/s13045-018-0645-x>

Sørensen, H. P., & Mortensen, K. K. (2005). Advanced genetic
strategies for recombinant protein expression in Escherichia
coli. *J Biotechnol*,*115*(2),
113-128. <https://doi.org/10.1016/j.jbiotec.2004.08.004>

Tchounwou, P. B., Yedjou, C. G., Patlolla, A. K., & Sutton, D. J.
(2012). Heavy metal toxicity and the environment. *Exp Suppl*,*101*, 133-164. <https://doi.org/10.1007/978-3-7643-8340-4_6>

Thirumoorthy, N., Manisenthil Kumar, K. T., Shyam Sundar, A.,
Panayappan, L., & Chatterjee, M. (2007). Metallothionein: an
overview. *World J Gastroenterol*,*13*(7),
993-996. <https://doi.org/10.3748/wjg.v13.i7.993>

Wu, Y., Li, H., & Li, Y. (2021). Biomineralization Induced by
Cells of Sporosarcina pasteurii: Mechanisms, Applications and
Challenges. *Microorganisms*,*9*(11). <https://doi.org/10.3390/microorganisms9112396>

Wu, Y., Li, X., Yu, L., Wang, T., Wang, J., & Liu, T. (2022).
Review of soil heavy metal pollution in China: Spatial distribution,
primary sources, and remediation alternatives. *Resources, Conservation and Recycling*,*181*, 106261. [https://doi.org/https://doi.org/10.1016/j.resconrec.2022.106261](https://doi.org/https%3A/doi.org/10.1016/j.resconrec.2022.106261)

Xu, X., Duan, L., Yu, J., Su, C., Li, J., Chen, D., Zhang, X., Song,
H., & Pan, Y. (2018). Characterization analysis and heavy
metal-binding properties of CsMTL3 in Escherichia coli. *FEBS Open Bio*,*8*(11), 1820-1829. <https://doi.org/10.1002/2211-5463.12520>

Yoon, J. H., Lee, K. C., Weiss, N., Kho, Y. H., Kang, K. H., &
Park, Y. H. (2001). Sporosarcina aquimarina sp. nov., a bacterium
isolated from seawater in Korea, and transfer of Bacillus globisporus
(Larkin and Stokes 1967), Bacillus psychrophilus (Nakamura 1984) and
Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as
Sporosarcina globispora comb. nov., Sporosarcina psychrophila comb.
nov. and Sporosarcina pasteurii comb. nov., and emended description of
th. *Int J Syst Evol Microbiol*,*51*(Pt 3),
1079-1086. <https://doi.org/10.1099/00207713-51-3-1079>

Yoshida, T., Yamaguchi, T., Iida, Y., & Nakayama, S. (2003). XPS
Study of Pb(II) Adsorption on γ-Al2O3 Surface at High pH
Conditions. *Journal of Nuclear Science and Technology*,*40*(9), 672-678. <https://doi.org/10.1080/18811248.2003.9715405>

You, J.-H., Song, B.-H., Kim, J.-G., Lee, M.-H., & Kim, S.-D.
(1995). Genetic Organization and Nucleotide Sequencing of the ure Gene
Cluster in Bacillus pasteurii. *Molecules and Cells*,*5*(4), 359-369. [https://doi.org/https://doi.org/10.1016/S1016-8478(23)17336-5](https://doi.org/https%3A/doi.org/10.1016/S1016-8478%2823%2917336-5)

Yu, R., He, L., Cai, R., Li, B., Li, Z., & Yang, K. (2017). Heavy
metal pollution and health risk in China. *Global Health Journal*,*1*(1), 47-55. [https://doi.org/https://doi.org/10.1016/S2414-6447(19)30059-4](https://doi.org/https%3A/doi.org/10.1016/S2414-6447%2819%2930059-4)

Zambelli, B., Stola, M., Musiani, F., De Vriendt, K., Samyn, B.,
Devreese, B., Van Beeumen, J., Turano, P., Dikiy, A., Bryant, D. A.,
& Ciurli, S. (2005). UreG, a chaperone in the urease assembly
process, is an intrinsically unstructured GTPase that specifically
binds Zn2+. *J Biol Chem*,*280*(6),
4684-4695. <https://doi.org/10.1074/jbc.M408483200>

Zeng, Y., Chen, Z., Du, Y., Lyu, Q., Yang, Z., Liu, Y., & Yan, Z.
(2021). Microbiologically induced calcite precipitation technology for
mineralizing lead and cadmium in landfill leachate. *J Environ Manage*,*296*, 113199. <https://doi.org/10.1016/j.jenvman.2021.113199>

Zhan-jun, K., Xin-zhe, L., Nan, L., & An-qing, G. (2018).
Ecological Risk Assessment of Heavy Metals in Surface Sediments in
Haiyan County, Zhejiang Province. *Journal of Ecology and Rural Environment*,*34*(12), 1096-1104.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}