iGEM 2024 — Team bit-china

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* [NOTEBOOK-bioleaching](#section1)
* [NOTEBOOK-biomineralization](#section2)
* [NOTEBOOK-biosorption](#section3)

# NOTEBOOK-bioleaching

# NOTEBOOK-biomineralization

# NOTEBOOK-biosorption

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* [Overview](#section2)
* [Bioleaching](#section3)
* [Biosorption](#section4)
* [Biomineralization](#section5)

# Overview

  "In engineering, failure is the mother of success. Understanding the reasons for failure is key to achieving success."

— Thomas Edison

“In engineering, failure is the mother of success.” This statement perfectly encapsulates
the essence of biological engineering, a discipline that continuously learns and grows from failure.

In the field of synthetic biology, scientists have solved a series of problems and grown from them through the rigorous application of learned
knowledge combined with practical situations. “Rooted in the known, embracing the unknown,” with a mindset like
this, innovation and creativity are being fostered continuously. We are excited to contribute to this cutting-edge
field as participants in iGEM. With perseverance and dedication, we will expand the possible boundaries of synthetic biology.

Our iGEM project focuses on the sustainable recovery of key metals such as Li, Co, Ni, and Mn from spent power batteries. With the three core
modules of bioleaching, bioadsorption, and biomineralization, our system continuously tackles challenges. These modules are constantly improved
in the iterative cycle of "design-construction-test-learn" to address increasingly complex challenges.

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage1.webp)

**Fig. 1** The iteration cycle of biological engineering

In the experimental design and implementation of the project, we conducted detailed testing and data analysis. Through the citric
acid produced by *Aspergillus niger* and the glucose oxidase from engineered Yeast, we optimized the efficiency of metal leaching and
employed the method of "Spent medium bioleaching" to enhance the leaching process's efficiency. In the bioadsorption section, we
utilized Yeast surface display technology to achieve the specific binding of metal ions. Finally, by expressing urease in an
*E. coli* system, we converted the captured metal ions into stable carbonates for the recovery and reuse of electrode materials.

Our experiments are conducted with extreme rigor.You can see the safety guidelines we follow on the
[**safety**](./safety.html) page, and you can browse the
standardized procedures we follow for our experiments on the [**protocol**](./protocol.html) page.
At the same time, we use a variety of ways to measure the results
of our experiments to explore the best [**measurement**](./measurement.html) criteria.
You can learn more about this in the [**result**](./result.html).

We have standardized all the components involved in our project to meet the requirements of the igem community and have
completed the part upload. You are more than welcome to use our [**part**](./parts.html) and contact us about its usage.
Any suggestions will be appreciated.

# Part1 Bioleaching

## Cycle 1: Design and Optimization of Leaching Solution1

### Design

**We selected citric acid as the primary leaching agent.** Considering that a single leaching acid may struggle to
adapt to the complex metal ion environment of black
powder leaching, we referred to the catalyst
recovery industry and initially **chose gluconic acid and hydrogen peroxide or ascorbic acid as supplementary
acids and reducing agents in the leaching solution.**

### Construction

During the leaching process, a series of leaching conditions will affect the outcomes of the leaching experiments.
Taking temperature as an example, excessively high temperatures can impact the activity of the leaching agent,
while appropriate heating can also enhance the reaction efficiency.

To explore the optimal leaching conditions for the leaching solution targeting black powder, we established a
series of gradient leaching experiments.

### Test

We conducted experiments in a round-bottom flask equipped with a spherical condenser.
Significant changes were observed during the experiment, **with the leachate gradually
transitioning from turbid to transparent**, indicating that the design of the leachate
was indeed effective.

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage2.webp)

**Fig. 2** Display of Leaching Effect

At the same time, based on a large amount of experimental data results , **we derived the optimal leaching parameters
for black powder: 70°C, 100 rpm, with a solid-liquid ratio of 15 g/L, using 1 M citric acid, 0.4 M gluconic acid, 0.6 M
hydrogen peroxide, or 0.25 M ascorbic acid (the leaching agent concentration can be scaled according to the solid-liquid ratio).**

### Learn

By conducting metal ion content detection on the extracted samples, we found that within the first half hour of the leaching
process, the samples were nearly fully leached, and the leaching rates of various elements could basically reach 80%-90%,
indicating that the leaching effect met expectations. After selecting the leaching agents, we next need to choose a suitable
microorganism to produce the substances we require.

## Cycle 2: Selection of leaching microorganisms

### Design

Initially, we selected *Aspergillus niger* as the producing strain for citric acid and gluconic acid,
as it is a commonly used strain in the industrial production of citric acid. To avoid imposing an excessive
metabolic burden on *Aspergillus niger*, **we induced the strain to produce citric acid in large quantities and
in a singular manner under low pH conditions.**

At the same time, we learned that the oxidation of glucose to gluconic acid is a one-step reaction catalyzed by glucose
oxidase (GOx), and the byproduct of this reaction is the reducing agent hydrogen peroxide that we require.

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage3.webp)

Ultimately, **we decided to utilize Yeast surface display technology, employing *Pichia pastoris* to display
GOx, and then co-cultivating the cultivated Yeast with a glucose-containing medium to obtain a large quantity of
gluconic acid.**

### Construction 1

In the cultivation of*Aspergillus niger*, based on Professor Jia's experience and preliminary literature research, we selected
the classic ME medium as the first-generation citric acid production medium, with the formulation of ME medium consisting of 30 g/L
malt extract and 5 g/L peptone.

### Construction 2

We utilized the Yeast surface display plasmid constructed in the bioadsorption section, employing the Gibson assembly method to
insert the GOx gene into it, and subsequently introduced it into *Pichia pastoris*, thereby obtaining a strain capable of surface
displaying glucose oxidase.

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage4.webp)

**Fig. 3** a) Design of GOx Plasmid; b) GOx Protein Gel Image

### Test 1

We experimentally verified that *Aspergillus niger* can rapidly proliferate in this medium; however, through subsequent
high-performance liquid chromatography (HPLC) analysis, the citric acid yield was only 2.38 mg/mL, which is far below the
literature yield of 50 mg/mL.

### Test 2

For the constructed engineered Yeast, we incubated it in a glucose-containing medium.
**Compared to the wild-type control group, the Yeast displaying GOx on its surface was able to lower the pH of
the medium more significantly.** This indicates that a reaction converting glucose to gluconic acid
occurred in the medium containing the engineered strain.

### Learn

The experiments showed that *Aspergillus niger* rapidly proliferated in the medium but the citric acid yield was
below expectations, while the engineered Yeast effectively converted glucose to gluconic acid in the glucose-containing
medium, indicating its potential in metabolic engineering. The next consideration is how to increase the citric acid yield.
After a more detailed literature review, we believe that improvements to the first-generation medium can be made from three
aspects: carbon source, pH, and nitrogen source, in order to enhance citric acid production.

## Cycle 3: Optimization of culture media

### Design

We optimized the medium for citric acid production by *Aspergillus niger*:
1) replacing the carbon source from malt extract to high-concentration glucose to promote
the large-scale production of citric acid; 2) adjusting the initial pH of the medium to
optimize citric acid production; 3) reducing nitrogen source supply by using sodium nitrate
instead of peptone to increase the carbon-to-nitrogen ratio, thereby inhibiting the growth of
*Aspergillus niger* and promoting citric acid production.

### Construction

Ultimately, the formulation of the second-generation medium was determined to be: 125 g/L glucose,
2 g/L NaNO3, 1 g/L K2HPO4, 0.5 g/L MgSO4·7H2O,
0.5 g/L KCl, and 0.01 g/L FeSO4.

### Test

We monitored the pH of the medium to characterize its growth status. Meanwhile, we used HPLC for quantitative
measurement of the citric acid content in the medium, and with the continuous improvement of the medium composition,
 **the citric acid yield showed a significant increase.**

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage5.webp)

**Fig. 4** a) HPLC Analysis of Citric Acid Concentration in
*Aspergillus niger* Culture Medium; b) Variation of Citric Acid Yield in Different Culture Media.

### Learn

From the results of the experiments, we have successfully achieved our expected goals and obtained a bioleaching
solution with a high citric acid content.

## Cycle 4: Selection of Leaching Method——Spent Medium

### Design

Considering the toxic effects of heavy metal ions contained in black powder on cells, the leaching rate of
One-step bioleaching is generally lower, although it shortens the cultivation time. We consider selecting a
more suitable method for the project through experiments between Two-step bioleaching and Spent medium bioleaching.

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage6.webp)

**Fig. 5** Schematic Diagram of Three Leaching Methods

### Construction

Two methods were used to leach the black powder under the same conditions, extracting small amounts of leachate at
gradient times to detect the metal ion content.

### Test

The results show that the medium method can complete leaching in about 30 minutes due to heating conditions, while the
two-step method takes several days. Surprisingly, the final leaching rates of both methods are not significantly different,
with the medium method having a slightly higher leaching rate than the two-step method, which contradicts our hypothesis.
We believe this may be due to the adsorption of metal ions by the mycelium, leading to many leached metal ions adhering to
the biomass surface, thus resulting in a lower calculated leaching rate of metal ions in the leachate. We conducted SEM-EDS
analysis on samples of *Aspergillus niger* before and after leaching, and **the results indicated the presence
of a large number of metal ions on the surface of *Aspergillus niger***, confirming our hypothesis .

### Learn

To maximize the leaching of free ionic metal forms quickly, **we chose the medium method based on our experimental results**.
In the bioleaching section, we mixed the culture broth of *Aspergillus niger* with GOx broth in the pre-experiment ratio and
leached the black powder using this method. We also need to determine an effective way to test *Aspergillus niger*'s leaching
efficiency for Li, Co, Ni, and Mn.

## Cycle 5: Iteration of characterization

### Design

We reviewed national standard detection methods for Li, Co, Ni, and Mn, including Spectrophotometry (SP), Atomic Absorption
Spectroscopy (AAS), and Energy Dispersive X-Ray Spectroscopy (EDX). **We selected AAS** because SP complicates the
experiment with color compounds and is prone to systematic errors, while EDX cannot detect lithium and is costlier. AAS, on the
other hand, enables automated continuous sampling with a robotic arm, reducing labor and time costs, and allows for the detection
of multiple elements by changing the hollow cathode lamp, enhancing convenience.

### Construction

We purchased hollow cathode lamps for the four elements Li, Co, Ni, and Mn, prepared standard solutions of these four
metal ions, and used them to plot the standard curves for the corresponding metal ions.

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage7.webp)

**Fig. 6** Concentration-Absorbance Standard Curve for the Four Elements

We used this standard curve to determine the metal content in the black powder and compared the obtained results
with the data provided by the seller. The error was within 10%, giving us reason to accept this standard curve. All metal ion detections in
this project were completed by AAS.

### Learn

The experimental world is not entirely consistent with what is presented in textbooks; while the former is a concretization
of theory, the latter is constrained and varied by countless real-world limitations and changes. Therefore, those seemingly
perfect theoretical methods often do not apply in practical operations. What is truly important may lie in the continuous
weighing, trial and error, and correction under these intricate conditions, ultimately finding a method that is not perfect
but sufficiently feasible, even capable of overcoming real-world constraints.

This pursuit belongs not only to the laboratory but also serves as a microcosm of life, teaching us how to find a reasonable
balance within limited conditions.

# Part 2 Biosorption

## Cycle 1: Selection and Prediction of Metal-Binding Peptides 1 ——Optimization of Polypeptide Geometric Structures and Molecular Mechanics Simulation

### Design

To accomplish the capture of metal ions, we conducted extensive literature research, and ultimately identified 10 types of
metal-binding peptides. **This includes one lithium-binding peptide (LBP3), two cobalt-binding peptides (CBP1/CBP2),
four nickel-binding peptides (NBP1/NBP2/NBP3/NBP4), and three manganese-binding peptides (mntR/BH2807/TssS).**

### Construction

Given the variable lengths of these peptides and the difficulty in meeting the peptide length requirements of Yeast surface
display technology, we have modified the peptide sequences. **We identified the interaction sites of the metal-binding
peptides with metals using the MIB2 tool, specifically the portion of the long peptides that effectively bind to metals,
and preserved that region while eliminating others**. For peptides that were too short, we duplicated their sequences
in order to improve their adsorption efficiency.

### Test

In order to assess the efficacy of the modified binding peptides, we utilized the calculation program MLatom from the XACS
platform to complete the structure calculations of the metal ion binding peptides with the metal ions. **We first
optimized the geometric structures of the polypeptide chains, then proceeded with the molecular mechanics simulation to
identify the bonding process of the polypeptides with the ions.**

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage8.webp)

**Fig. 7** Preliminary Structural Optimization of Select MBPs (Metal-Binding Peptides)

### Learn:

In this round of engineering, we obtained metal-binding peptides that are suitable for Yeast surface display and verified their
binding effects on metal ions, enabling us to proceed with the next steps of the experiment.

## Cycle 2: Selection and Prediction of Metal-Binding Peptides - Optimization of Polypeptide Model Prediction and ITC Detection

### Design:

Through structural optimization using machine learning algorithms, we often obtained unreasonable structures, such as cases of
covalent bond breaks, non-converging SCF calculations, and potentially meaningless computation results. Therefore, we communicated
with Professor Shilu Chen(For more details, please refer to [**HP**](./human-practices.html))from the School of Chemistry
and Chemical Engineering and adjusted the input files according to his suggestions to obtain more accurate results for the structural
optimization of the metal-binding peptides.

### Construction:

Firstly, **we confirmed the accurate connection of the amino acid chains**, ensuring the use of L-amino acids and that no hydrogen atoms were
lost. When adjusting the peptide chain structures, we ensured that they were chemically reasonable, avoiding any overlapping or excessively
close atoms. This is because machine learning methods, when applied, do not consider bond connections but atom distances. Thus, any errors
in bond judgments during optimization might lead to erroneous decomposition and incorrect results. Given that the polypeptides might bear
charges, it is important to consider the environmental pH and proton transfer, and it is not necessary to insist on counterion addition.
Adding too many small molecules during structure optimization increases the state complexity and could have unforeseen negative effects.
Moreover, **we also calculated the binding energies of some metal-binding peptides to four types of metals in order to assess the specificity
of their adsorption.**

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage9.webp)

**Fig. 8** Iterative Structural Optimization of MBPs (Short Peptides)

**Table 1** inding Energy of Select MBPs with Four Types of Metal Ions

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage10.webp)

### Test

When calculating the binding energy of some metal-binding peptides with four types of metals, we found the outcome for the short
peptide NBP4 was not ideal, although its molecular mechanics simulation performance was decent. To evaluate NBP4's binding effect
with metal ions, we employed Isothermal Titration Calorimetry (ITC) to characterize its binding capacity to the corresponding
target ions and conducted binding experiments with other ions. The isothermal titration calorimetry experiment demonstrated that
the cyclic nickel-binding peptide, with a sequence of CNAKHHPRCGGG, forms a complex with Ni2+ ions. However, no interaction was d
etected between the nickel-binding peptide and Li+, Mn2+, and Co2+. This nickel-specific peptide selectively
binds with nickel.

### Learn

Initial structure optimization serves as the basis for obtaining reasonable subsequent results. **By enhancing our peptide structure optimization approach, we
achieved improved results. Calculating the binding energy of peptides to metal ions enables us to intuitively assess their efficacy. Employing ITC to measure
the binding affinity of peptides to metal ions, we verified and further supported the model's predictions**, thereby providing a solid basis for our subsequent
experimental endeavors.

## Cycle 3: Selection of Adsorption Strategy: Construction of Engineered Yeast Displaying MBPs

### Design

To express the optimized metal-binding peptide on the Yeast surface, we first constructed an efficient surface display plasmid using
*Pichia pastoris* as the expression vector. The backbone sequence pir1 links to the target protein via a linker, anchoring it
on the Yeast surface, while the signal peptide α-factor initiates secretion. We also included the bleomycin resistance gene in the plasmid
to aid in screening transformed strains.

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/e9.webp)

**Fig. 9** Construction of the surface display plasmid pGAPZaA-pir1.

### Construction

After constructing the plasmid's foundational part via primer PCR, we inserted different MBP genes to create distinct surface display
plasmids, using the Gibson assembly method for long peptide genes. We then introduced these plasmids into *E. coli* for amplification
and extracted them afterward. After sequencing and confirming the plasmids, we transformed them into *Pichia pastoris* using
electroporation. Finally, we verified the successful introduction of the plasmids into *Pichia pastoris* through colony PCR,
resulting in engineered Yeast displaying MBPs on their surface.

### Test 1 Fluorescence microscopy observation:

We used an ELISA-based method for immunofluorescence detection due to the inclusion of a Myc tag in the surface plasmid. Successful
cell surface display was indicated by distinct red fluorescence, while its absence suggested failure.**The results showed no
fluorescence in the control group, whereas the experimental group exhibited strong fluorescence with a halo around the cell edges,
confirming successful surface display.**

### Test 2 Graphite furnace detection:

To assess adsorption efficacy, we mixed the engineered bacterial suspension with prepared single-metal solutions in a specific ratio.
After 2 hours, we centrifuged the mixture to obtain the supernatant and measured the concentration of metal ions using a graphite furnace.
**Calculations showed that the engineered bacterial strains had a certain level of adsorption for the target metal ions.**

### Test 3 Scanning Electron Microscopy(SEM) Observation:

The bacterial precipitates obtained after adsorption and centrifugation were used for sample preparation and observation by SEM.
We observed that the cells in the control group (without the surface display plasmid) had smooth surfaces, while the cells in the
experimental group (with the surface display plasmid) had rougher surfaces. This difference is likely attributed to the surface display
proteins.

### Learn

We have learned and practiced how to construct effective plasmids for surface display, and successfully obtained multiple strains
capable of displaying different metal-binding peptides. Their effectiveness has also been verified. However, only a few strains exhibit
satisfactory adsorption effects. For industrial applications, the adsorption efficiency of the engineered strains needs to be improved.

## Cycle 4: Selection of Adsorption Strategy - Self-assembling of NBP4 (Nickel-Binding Peptide 4)

### Design

The binding sites of metal ion-binding peptides in their monomeric form are limited, while self-assembling peptides can form
various binding sites. Therefore, **we attempted to enhance the binding efficiency of metal ion-binding peptides through a
self-assembly strategy**. We tried AlphaFold3 to predict the structures of all metal-binding peptides involved in the adsorption
process and found that the 32-mer structure of N4 exhibited potential for self-assembly. Thus, we decided to conduct further research
on this.

### Construction 1

After conducting a literature review, we adopted three temperature gradients: 4°C, 25°C, and 37°C, to investigate their respective
effects on self-assembly. Microstructures were observed at 24 h and 48 h to determine the optimal temperature and time for self-assembly.

### Construction 2

Considering the specific binding affinity of the imidazole group in the R-group of histidine for metal ions, we used catalytic
amounts of metal ions to induce the self-assembly of N4 into regular spherical structures. We selected four common metal ions,
Ni2+, Mn2+, Co2+, and Ca2+, all of which are divalent metal ions with coordination ability.

### Construction 3

We co-incubated the self-assembled N4 with NBP4-pir Yeast strains, followed by oscillation culture with Ni2+ solution.
Ultimately, EDS analysis was employed to measure the nickel content on the Yeast cell surface, aiming to verify the enhancement
effect of self-assembly on adsorption. Concurrently, we also designed a cell-free experiment to observe the reaction between the
peptide solution, which had not been adsorbed by Yeast, and nickel ions, and scanned their nickel content using EDS.

### Test 1

For each group of samples, we observed the morphologies of self-assembly at both 24 h and 48 h. It was evident that at 48 h, the
self-assemblies were spherical and generally larger and more uniform than those formed at 24 h. When samples were collected at 24 h,
only the group incubated at 37°C exhibited microspheres. At 48 h, all three groups displayed spherical self-assembled peptides, with
the group at 37°C showing a higher peptide density and considerable size.

### Test 2

We added four types of metal ions separately and observed their morphologies after self-assembly for 24 h and 48 h at 37°C.
Concurrently, we measured the particle size and distribution of the self-assembled peptide spheres at 48 h. Through AFM
characterization experiments, we could clearly observe from the images that, under the condition of 37°C and with a catalytic
amount of Ni, N4 formed a large number of uniform spherical assemblies after 48 hours of assembly. Among the various metal ions
tested for induction, **Ni exhibited the most effective induction effect**, independently verifying the specific
binding ability of N4 to Ni.

### Test 3

In the co-incubation experiments with assembled cells, we observed a significant difference in Ni content between the Yeast
surface and the substrate, confirming that the Yeast surface was loaded with peptide assemblies enriched in Ni. In contrast,
in the group where monomers were co-incubated with cells, high concentrations of Ni were almost undetectable on the cell surface.

### Learn

We successfully achieved self-assembly of N4 and identified the optimal time, temperature, and catalyst for well-assembled N4.
We verified that the assemblies could bind to cells and adsorb significant amounts of nickel ions, demonstrating their advantages
over monomers. In the future, we will continue to explore self-assembly strategies and apply them to other engineered strains to
enhance their adsorption capabilities.

# Part3 Biomineralization

## Cycle 1: Selection of Mineralization Method 1 — Surface Display of SazCA in *E. coli* BL21 (DE3) for Biomineralization

### Design

SazCA is recognized as one of the fastest known carbonic anhydrases and **will be expressed through genetic engineering by
fusing it with the outer membrane protein INPN of *E. coli*, allowing it to be exposed to the extracellular environment**.
This enzyme efficiently catalyzes the hydration of CO2 to generate bicarbonate, thereby facilitating the formation of
carbonate minerals. The design emphasizes improving the contact efficiency between the enzyme and the substrate, with the expectation
of enhancing mineralization efficiency.

### Construction

The gene constructs and plasmids developed for the surface display of SazCA are as follows:

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage12.webp)

**Fig. 10** Schematic representation of gene routes displayed on the surface of SazCA

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage13.webp)

**Fig. 11** SazCA surface display plasmid construct

### Test

Introduce the plasmid obtained from the company into BL21(DE3), followed by a low-temperature induction culture in a shaking incubator
at 0.4 mM IPTG, 0.5 mM ZnSO4, 25 °C, and 220 rpm for 24 hours. After centrifugation, resuspend the cells in a 0.02M Tris-HCl
buffer solution at pH 8.3, then add a cold saturated solution of carbon dioxide and react on ice for 4 hours. Collect the supernatant
(initial pH of 5.35) and incorporate it into the mineralization system detailed in the table below. Incubate at room temperature (25 °C)
for 12 hours to observe the mineralization results.

**Table2**  Configuration of the SazCA mineralization system

|  |  | Configuration of the SazCA mineralization system |  |  |
| --- | --- | --- | --- | --- |
| Ca（The final concentration is 0.5M） | Li（The final concentration is 0.2M） | Mn（The final concentration is 0.15M） | Ni（The final concentration is 0.5M） | Co（The final concentration is 0.5M） |
| 3mL supernatant +3mL 1M CaCl2 | 3mL supernatant +3mL 400mM LiCl | 3mL supernatant +3mL 0.3M MnCl2 | 3mL supernatant +3mL 1M NiCl2 | 3mL supernatant +3mL 1M CoCl2 |

However, the results were not as anticipated; no precipitation was observed in any of the five tubes.

### Learn

Upon further investigation, we concluded that carbonate mineralization is more effective under alkaline conditions,
where carbonate ions (CO₃²⁻) are stable and promote mineral phase formation. However, the carbonic acid (H₂CO₃) produced
from the reaction of SazCA with carbon dioxide in water creates a mildly acidic environment. This acidity reduces the
concentration of available carbonate ions and hinders stable mineral phase nucleation, negatively impacting mineralization efficiency.

## Cycle 2: Selection of Mineralization Method 2 — Recombinant Expression of Urease from *S. pasteurii* in *E. coli* and Validation of Urease Activity

### Design

Due to the detrimental acidic environment created by surface-expressed SazCA on metal ion mineralization, we turned to the urease gene from *S. pasteurii* for recombinant expression in *E. coli*. Our transition to using *S. pasteurii* urease for biominization studies was guided by the following considerations:

1.Environmental Adaptability: *S. pasteurii* thrives in alkaline conditions, where its urease is highly active, aligning with our mineralization needs.

2.Biological Principles: The hydrolysis of urea by *S. pasteurii* urease generates carbonate ions and raises pH, favoring carbonate mineral formation.

3.Previous Research: Studies show that *S. pasteurii* urease has diverse applications in biominization, such as concrete repair and heavy metal stabilization, providing valuable insights.

Our goal is to enhance biominization under mildly alkaline conditions to improve the synthesis efficiency of Li, Mn, Co, and Ni carbonates. We will quantitatively assess urease activity by measuring enzyme expression levels and aim to optimize key factors to boost urease activity.

### Construction 1

Due to the large size of the efficient urease gene from *S. pasteurii*, we divided it into two segments, which were constructed on the corresponding plasmids. UreA, UreB, and UreC were expressed using the pET28a vector (T7 promoter), while UreE, UreF, UreG, and UreD were expressed using the pET21b vector (proD promoter).

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage14.webp)

**Fig. 12** pET28a (up-containing genes UreA, UreB, UreC) (left);
pET21b (upper containing genes UreE, UreF, UreG, UreD) (right)

### Construction 2

The Urease (UE) Activity Assay Kit was purchased from Boxbio for the quantitative detection of urease activity.

### Construction 3

Furthermore, we utilized SDS-PAGE to run protein gels in order to assess the molecular weights of the subunits of urease and determine whether the recombinant urease subunits were expressed correctly.

### Construction 4

Furthermore, we utilized SDS-PAGE to run protein gels in order to assess the molecular weights of the subunits of urease and determine whether the recombinant urease subunits were expressed correctly.

### Test 1 Construction and Expression of Urease Gene Dual Plasmid System

In a laminar flow hood, TOP10 competent cells were mixed with the plasmids, followed by heat shock and ice bath treatments. LB medium was then added for shaking culture, and subsequently, the mixture was plated on labeled agar plates for transformation experiments.

### Test 2 Quantitative Detection of Urease Activity

Based on the calculations from the Urease (UE) Activity Assay Kit instructions, the urease activity was found to be low (with minimal difference from the control group), indicating that issues occurred during the recombinant expression process, leading to reduced activity. We will further analyze the expression of urease subunits using SDS-PAGE.

### Test3 SDS-PAGE

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage15.webp)

**Fig. 13** SDS-PAGE of dual plasmids pET21b and pET28a (where UreC: 61.4 kD; UreD: 29.3 kD;
UreG: 23.1 kD; UreF: 23.0 kD; UreE: 17.4 kD; UreB: 14.0 kD; UreA: 11.1 kD)

The SDS-PAGE conducted with whole-cell lysate displayed a complex pattern, with bands not clearly distinguishable. Although some corresponding bands were observed, the protein bands were faint, indicating poor translation and expression of the subunits.

### Learn

Here, we conducted a preliminary analysis of the activity of the recombinant urease. Two main issues were identified concerning SDS-PAGE: first, the faint bands of the subunits UreE, UreF, UreG, and UreD on the pET21b plasmid, which indirectly suggest a low concentration of the corresponding proteins. The recommendation is to replace the proD promoter with the T7 strong promoter to enhance the expression of downstream genes, as well as to optimize the induction conditions.

## Cycle 3: Optimization of *S. pasteurii* Urease Recombinant Expression in *E. coli*

### Design

Based on the issues encountered in Cycle 2 regarding urease expression, we planned the following improvements: replacing the promoter by substituting the proD promoter preceding UreE, UreF, UreG, and UreD with the T7 strong promoter to enhance downstream gene expression. Additionally, we will optimize the induction conditions, which primarily include IPTG and Ni2+ concentrations, as well as the temperature and duration of induction, to improve the activity of urease expression.

### Construction

In this phase, we used the laboratory plasmid pET21a (which contains the T7 promoter) to design primers for PCR and reverse PCR to linearize the target gene and vector separately. Subsequently, we performed Gibson assembly to reconstruct the plasmid, aiming to replace the proD promoter with the T7 strong promoter preceding UreE, UreF, UreG, and UreD.

### Test 1 Quantitative Detection of Urease Activity

Upon receipt of the primers, we proceeded with PCR and reverse PCR to linearize the target gene and vector. After gel purification and genomic DNA purification, we performed Gibson assembly to reconstruct the plasmid. The assembled DNA was then transformed into host cells for amplification and colony PCR screening, retaining only those strains with favorable sequencing results for plasmid extraction and dual plasmid transformation. After extensive literature research, **we determined that inducing under the conditions of 0.5 mM IPTG, 5 µM NiCl₂, 20℃, and 220 rpm for approximately 16 hours** would be more beneficial for urease expression. Subsequent SDS-PAGE analysis of the urease subunit expression showed darker bands, indicating significant improvement in the translation of urease subunits.

### Test 2

Absorbance measurement: The absorbance at 630 nm was recorded as A\_test (dual plasmids) and A\_control (BL21). Substituting values into the standard curve from Cycle 2 Test 2 for urease activity quantification yielded **an activity of 0.153 U/g DCW** (U: the amount of enzyme that catalyzes the formation of 1.0 µmole of ammonia per minute at pH 7.0). Compared to Cycle 2 Test 2, urease activity was improved.

### Learn

Through the experiments outlined above, we improved the recombinant expression of urease and demonstrated that the recombinant urease possesses activity. Next, we will utilize the optimized recombinant expression strain for the biocalcification of Li, Mn, Co, and Ni.

## Cycle 4: Evaluation of *S. pasteurii* Urease Recombinant Expression and Mineralization in *E. coli*

### Design

After confirming the activity of urease, we proceeded to investigate the biocalcification of four metal ions: Li, Mn, Co, Ni, and Ca (where CaCO₃ is the most common biocalcification product induced by urease, used here for a series of experimental controls). Demonstrating that our recombinant urease can effectively decompose urea to generate sufficient carbonate ions that combine with metal ions to form the corresponding carbonates is a process we need to explore continuously. Based on preliminary visual observations, we plan to first use a light microscope to examine the impact of mineralization on bacterial growth. Additionally, **we will use Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) to compare the surface morphology of the bacteria and analyze the elemental composition and content on the bacterial surface** (higher metal ion content will indicate improved adsorption and mineralization).

### Construction

We expanded the seed culture and induced under the conditions based on the findings from Cycle 3 Test 1. Following this, we performed biocalcification and assessed the mineralization results. Before conducting SEM and EDS analyses, we plan to perform a preliminary examination of the mineralization results using a light microscope: we will take precipitates from all mineralization systems, dilute them appropriately in deionized water, and place 1 µL on a microscope slide for observation.

### Test 1 Optical Microscope Observation

Observations using the optical microscope indicated that, compared to the BL21(DE3) control group, the dual plasmid experimental group of recombinant urease showed significant differences in the mineralization of Li, Mn, Co, and Ni: it was evident that the bacteria formed clumps and were surrounded by a distinct layer of material.

### Test 2 Confocal Microscope Observation

To further explore whether the material attached to the bacteria in the experimental group was the corresponding carbonate mineralization product, we conducted comparative observations using a Confocal Microscope.

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage16.webp)

**Fig. 14** Results of biocalcification observed by Confocal Microscope:
a) Control group of Ca; b) Control group of Li; c) Control group of Mn; d) Control group of Ni; e) Control group of Co;
f) Experimental group of Ca; g) Experimental group of Li; h) Experimental group of Mn; i) Experimental group of Ni; j) Experimental group of Co

However, during the observations using the Confocal Microscope, we were unable to zoom in on individual bacteria to examine the surface characteristics. Although the experimental group displayed noticeably darker shadows and clumping phenomena compared to the control group under the same parameters, this still does not provide strong evidence for our mineralization phenomenon.

### Test 3 Polarized Light Microscope Observation

After further consultation with Professor Ma Yurong(details available on [**HP**](./human-practices.html)), she recommended that we conduct observations using a polarized light microscope. This is due to the unique crystal structure of carbonate minerals, which produces a birefringence phenomenon, resulting in distinct optical features such as interference colors and extinction positions. These characteristics will assist us in determining whether the surface of our mineralization products is coated with carbonate minerals.

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage17.webp)

**Fig. 15** Observation of biocalcification results via Polarized Light Microscopy.
(left: dark stripes; right: light stripes)

Calcium carbonate crystals appear white under dark stripes and black under light stripes. However, our biocalcification samples showed no crystal birefringence under dark stripes in the polarized light microscope. Nickel carbonate and cobalt carbonate also lacked crystal characteristics under this microscopy. After consulting Professor Long, we learned that some crystals, like silica, do not display luminous phenomena under dark field illumination. This indicates that polarized light microscopy may not accurately represent our mineralization results, possibly due to the need for sample transparency or semi-transparency, which our biocalcification samples do not possess.

### Test 4 Atomic Force Microscopy (AFM) Observation

During our literature review, we found that **Atomic Force Microscopy (AFM) is considered an ideal tool for observing biocalcification
results due to its high resolution**, three-dimensional imaging capabilities, and non-destructive nature. It can reveal structural
details of biominerals at the nanoscale, measure mechanical properties, and monitor the mineralization process in real time, providing
intuitive and precise data for understanding the mechanisms of biomineralization. Therefore, we attempted to analyze the surface morphology
and height of the bacteria using AFM to evaluate our mineralization results. (Note: Initially, we performed AFM observations on the
mineralization of the double plasmid experimental group Ca and the control group to preliminarily assess the feasibility of AFM for
mineralization analysis. For details on AFM analysis of Li, Mn, Co, and Ni, please refer to the [**results**](./result.html) section.)

The results are as follows:

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage18.webp)

**Fig. 16** AFM Observation of Biocalcification Results a) Ca Double-Plasmid Experimental Group;
b) BL21 Control Group

It is evident that the bacteria in the control group are relatively dispersed, with a flatter surrounding environment. In contrast, the
double-plasmid experimental group shows a noticeable aggregation of bacteria, accompanied by increased height in the surrounding area, which
further confirms the presence of some adhered substances around the bacteria. Next, we will conduct SEM and EDS analyses to evaluate our
mineralization results by comparing the surface morphology of the bacteria and analyzing the elemental composition and content on the bacterial
surface (higher metal ion content indicates better adsorption and mineralization of metal ions by the recombinant *E. coli*).

### Test 5 SEM and EDS Analysis

Given that calcium carbonate biomineralization is the most common, we will first verify the mineralization level of recombinant urease using Ca2+ as an example. For details on the SEM and EDS analyses of Li, Mn, Co, and Ni, please refer to the results section. (Note: Due to the limitations of scanning electron microscopy, elements with an atomic number below that of carbon cannot be analyzed using EDS; therefore, Li cannot be detected with EDS.)

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage19.webp)

**Fig. 17** SEM images of Ca2+ biomineralization results (Note: Negative control
is biomineralization by co-incubation with metal ions using wild-type BL21 bacteria without urease double plasmid; Positive control
is simulated in vitro biomineralization by adding urease in addition to wild-type BL21 bacteria; Experimental group is the
biomineralization of BL21 bacteria with recombinantly expressed double plasmid containing urease in the present project)
a) Electromicrograph of Ca negative control; b) Electromicrograph of Ca Electron micrograph of experimental group; c) Electron
micrograph of Ca positive control

![Project Flow Diagram](https://static.igem.wiki/teams/5166/images/engineering/eimage20.webp)

**Fig. 18** EDS analysis of Ca. a)Schematic diagram of Ca carbonate biomineralization;b)EDS layered images of Ca;c)EDS analysis of Ca negative control;d)EDS analysis of the Ca
experimental group;e)EDS analysis of Ca positive control

As shown in the figure above, **the Ca2+ experimental group and the positive control exhibited significant mineralization**.
In the experimental group, the bacterial cells display swelling at one end or the center, resulting in a club-like morphology.
The positive control group formed hemispherical carbonate precipitates around the bacterial cells, with more pronounced intercellular adhesion.
In contrast, the surface of the negative control bacteria appears relatively smooth, showing no signs of aggregation. Furthermore, EDS results
reveal that the Ca content analysis yields the following order:**positive control > experimental group > negative control**. These results are
consistent and relatively favorable, providing substantial evidence that the recombinant urease is active and that it can adsorb more Ca2+
compared to the negative control, effectively facilitating calcium carbonate biomineralization. This capability lays the groundwork for the
subsequent biomineralization of Li, Mn, Co, and Ni.

### Learn

Through a series of validation tests on the mineralization results, we have preliminarily demonstrated that urease from *S. pasteurii* can be successfully expressed recombinantly in *Escherichia coli* and can effectively facilitate mineralization. However, there remains considerable scope for improvement. Moving forward, our focus will be on enhancing urease activity and further optimizing mineralization conditions to increase the efficiency of biomineralization. Additionally, how to effectively repurpose carbonic anhydrase presents a topic worthy of exploration, particularly given the significant potential of SazCA in CO2 neutralization. To enable the practical application of SazCA, future research should focus on optimizing its activity and expression based on protein sequences and genetic circuits. This includes designing reaction conditions tailored to specific application environments and developing coupled synergistic systems within upstream and downstream metabolic processes(For more information, please see [**implementation**](./implementation.html)).

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