iGEM 2025 — Team cug-china

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![](https://static.igem.wiki/teams/5665/first-photo/f-notebook.webp)

Diary of wet experiment

## Preparation：

The team members conducted extensive literature reviews and studied previous iGEM projects while brainstorming ideas for the 2025 project. Eventually, they discussed and determined the general direction and framework of the project.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/nb1.webp)

They discussed the project’s feasibility with the PI and advisors and further refined the project details.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/nb2.webp)

## Week 1 (May 20–May 28)

This week, we acclimated *A. ferrooxidans* using glucose, determined the growth curve and ferrous ion concentration of *A. ferrooxidans*, and constructed a microbial electrochemical device based on wild-type *A. ferrooxidans*. Unfortunately, both two were unsuccessful.

## Week 2 (May 29–June 6)

This week, we determined the growth curve and ferrous ion concentration of the wild-type *A. ferrooxidans*. Based on the results of previous experiments, we adjusted and optimized the experimental protocol. Reconstructed the microbial electrochemical device based on wild-type *A. ferrooxidans*, as the initial experiment failed due to a short circuit caused by a broken connection between the wires and the electrode.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/nb3.webp)

## Week 3 (June 7–June 14)

We completed the restriction enzyme site analysis and primer adaptation of the pYDT vector and the Ptac promoter.And we prepared YPD medium, performed the plate-pouring operation for YPD and LB media, carried out streak isolation of *Saccharomyces cerevisiae*, and picked single colonies of *E. coli DH5α* 0053. Reconstructed the microbial electrochemical device using wild-type *A. ferrooxidans* . After two weeks of stable operation, we achieved preliminary success and obtained data results from the wild-type microbial electrochemical system.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/nb4.webp)

## Week 4 (June 14–June 21)

This week, we extracted the genome of *A. ferrooxidans*, picked single colonies of PYDT, and extracted the plasmid from *E. coli DH5α 0053*.

## Week 5 (June 22–June 28)

We constructed bioelectrochemical devices for the engineered strains pYDT and p0053. After two weeks of operation, Module I ran successfully, and we obtained the expected results.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/nb5.webp)

## Week 6 (June 29–July 5)

Using the genomic DNA of *A. ferrooxidans* as a template, we amplified the upstream fragment of the AFE\_0536 gene via PCR. After gel extraction, the fragment was stored at low temperature for subsequent use.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/nb6.webp)

## Week 7 (July 6–July 14)

We amplified the downstream fragment of the AFE\_0536 gene via PCR. After gel extraction, the downstream fragment was subjected to the first round of overlap extension PCR (OE-PCR) together with the upstream fragment obtained last week.

## Week 8 (July 15–July 21)

Using the product of the first round of OE -PCR as a template, we conducted the second round of overlap extension with full-length primers to obtain the complete AFE\_0536 gene fragment, which was then purified by gel extraction.

## Week 9 (July 22–July 28)

The AFE\_0536 fragment and the pYDT vector were double-digested with restriction enzymes, followed by overnight ligation using T4 DNA ligase. The ligation product was transformed into*E. coli DH5α* competent cells, and the transformed cells were spread on LB agar plates (supplemented with 50 μg·mL⁻¹ streptomycin) for overnight cultivation.

## Week 10 (July 29–August 5)

The pYDT-Ptac-AFE\_0536 plasmid with correct sequencing results was extracted, quantified using a NanoDrop spectrophotometer, and stored at −20 ℃. Meanwhile, after streak plating, single colonies were picked, cultured in a shaker for one day, and then the bacterial culture was preserved.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/nb7.webp)

## Week 12 (August 14–August 21)

The constructed plasmid was transformed into the target expression host. Positive clones were re-verified by PCR, and 20% glycerol stocks of the positive clones were prepared. Subsequent expression verification was initiated.

## Week 13 (August 22–August 28)

This week, we continued to determine the growth curves of *A. ferrooxidans*and *E. coli*. We focused on comparing the changes in OD₆₀₀ values of different strains (such as PYD1, Series F, WT, etc.) at different time points to evaluate their growth status.

## Week 14 (August 29–September 5)

We conducted experiments to determine the ferrous ion concentration, and initially explored the utilization of ferrous ions by the strains under different treatment conditions, providing data support for the subsequent optimization of culture conditions.

## Week 15 (September 6–September 12)

This week, the focus of the experiment shifted to comparing the growth performance of *A. ferrooxidans* and*E. coli*BL21 in different media (such as LB and M9), and we continued to determine the changing trends of their OD₆₀₀ values. Meanwhile, we further carried out experiments to determine the ferrous ion concentration to evaluate the ferrous oxidation capacity of the strains at different time points. By comparing the growth curves and ferrous ion utilization efficiency of different strains, we provided a basis for the subsequent screening of high-efficiency strains and optimization of experimental conditions.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/nb8.webp)

## Week 16 (September 13–September 20)

This week, we systematically determined the growth curve (monitored by OD₆₀₀) and ferrous oxidation capacity (monitored by OD₅₆₂) of *A. ferrooxidans*  at different time points. The experimental setup included different dilution factors (2-fold, 4-fold, and undiluted) and multiple time points (0 h, 12 h, 24 h, 36 h, 48 h).

## Week 17 (September 21–September 28)

This week, we systematically determined the growth curve (monitored by OD₆₀₀) of *E. coli*BL21 at different time points. The experimental setup included different dilution factors (2-fold, 4-fold, and undiluted) and multiple time points (0 h, 12 h, 24 h, 36 h, 48 h).

## Week 18 (September 29–October 6)

We organized the experimental records, original electrophoresis images, sequencing reports, and plasmid maps. Plasmids, glycerol stocks, primer information, and restriction enzyme digestion identification images were archived and stored in the warehouse. We completed the dual filing of paper and electronic data in the laboratory, preparing complete materials for subsequent protein expression and functional verification.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/nb9.webp)

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![](https://static.igem.wiki/teams/5665/first-photo/f-project.webp)

Engineering

CYCLE ONE

CYCLE TWO

CYCLE THREE

CYCLE FOUR

CYCLE FIVE

Our project

References

Design and Implementation of a First-Generation Microbial Electrochemical System Based on *Acidithiobacillus ferrooxidans*

## Design

*A. ferrooxidans* is capable of obtaining energy by oxidizing ferrous iron (Fe²⁺) or reduced sulfur compounds (such as sulfide, elemental sulfur, and thiosulfate) in acidic environments with a pH of 1.5–2.5. Electron transfer plays a crucial role in the cellular activities of *A. ferrooxidans*. Iron, a multivalent metal, exists in solution as either Fe²⁺ or Fe³⁺ and can act as an effective electron shuttle. It has been reported that *A. ferrooxidans* utilizes soluble iron as an electron shuttle[1].

Based on these findings, we propose to employ ***A. ferrooxidans***  as a chassis organism to construct a **microbial electrochemical** system and explore its potential electrochemical applications.

## Build

Unlike typical electroactive microorganisms such as *Shewanella* and *Geobacter*, which are commonly used in microbial fuel cells (MFCs), *A. ferrooxidans* exhibits a distinct electron transfer pathway during the oxidation of ferrous iron: electrons are transferred from the extracellular environment into the cell.

In conventional MFCs, electricity is generated spontaneously through outward electron transfer from cells to an anode. In contrast, an *A.ferrooxidans*-based microbial electrochemical system functions as a microbial electrolysis cell (MEC) that consumes electrical energy to drive biochemical reactions[2].

Based on the principles of microbial electrolysis, we have designed a system that utilizes electrons derived from ferrous iron oxidation to power CO₂ fixation and biomass production. In this configuration, electrons flow from the cathode to reduce ferric ions (Fe³⁺) to ferrous ions (Fe²⁺). *A. ferrooxidans* re-oxidizes Fe²⁺ back to Fe³⁺, completing the iron cycle while enabling carbon fixation.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f1-1.webp)

**A three-electrode system** was assembled in a sterile reactor, with both the cathode (1 cm×3 cm = 3 cm²) and anode (3 cm×4 cm =12 cm²) made of hydrophobic carbon cloth. The cathode potential was maintained at -0.2 V vs. Ag/AgCl (saturated KCl, E° = 0.197 V vs. SHE), which served as the reference electrode. The reactor consisted of a 100 mL glass bottle sealed with a perforated rubber stopper and a breathable membrane to secure the electrodes while allowing gas exchange and maintaining sterility. Electrical connections were established using wire as the current collector.

The electrolyte was 9K medium supplemented with ferrous iron and adjusted to pH = 2. To minimize Fe³⁺ precipitation and enhance current output, 9 mM sodium citrate (C₆H₅Na₃O₇·2H₂O) was added.

*A. ferrooxidans* was pre-cultured in a 250 mL flask containing 150 mL of 9K medium (44.22 g/L FeSO₄·7H₂O) at 30°C with shaking at 150 rpm. Cells were harvested by centrifugation at 5500 rpm for 5 minutes, resuspended in fresh medium, and inoculated into the reactor at an OD600 of 0.1. The electrodes were connected to a potentiostat, and **current–time curves** were recorded to monitor the electrochemical activity of the engineered strain. The reactor was incubated at 30°C in a constant-temperature incubator. Detailed medium formulations and operational procedures are provided in the **protocol** section.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f1-2.webp)

Figure 1-2 Microbial electrochemical reactor based on the initial construction of *A. ferrooxidans*

## Test

To investigate the capability of **wild-type***A. ferrooxidans* to utilize electrons, we recorded the **current-time curves** and calculated the current density based on the working electrode area.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f1-3.webp)

Figure 1-3 Initial build of *A. ferrooxidans*-based reactor with detected current-time curves (i1, i2 and i3 are parallel controls)

Theoretically, the current-time curve was expected to exhibit**a declining trend** due to the depletion of ferrous iron in the medium and limitations in cell growth. However, the collected data did not reflect this anticipated decrease. More critically, shortly after connecting the system to the potentiostat, the recorded current dropped to zero. Subsequent inspection identified a broken connection between the carbon cloth electrode and the current collector wire as the root cause.

## Learn

The observed failure can reasonably be attributed to metal corrosion in the extremely acidic electrolyte (pH = 2). **This highlights a central paradox in working with *A. ferrooxidans*: its acidophily is both an advantage and a challenge.** From a synthetic biology perspective, the extreme acidity creates a selective environment where most contaminating microbes cannot thrive, thereby reducing the need for costly antibiotics. However, this same condition demands that all experimental materials, including reactor vessels and electrical contacts, must be highly acid-resistant.

**The broader implications of this acidic environment were evident in our 2024 project.**
Although the *E. coli* *yedQ* gene is a highly efficient diguanylate cyclase (DGC) in other hosts, its expression and enzymatic activity in*A. ferrooxidans*  were suboptimal compared to native DGCs, likely due to the physiological constraints of extreme acidophily. Consequently, a critical re-evaluation of our current collector material is imperative for the success of any electrochemical application.

Construction of a Microbial Electrochemical System Based on *A. ferrooxidans*

## Design

Based on our literature review, we selected a titanium wire with a diameter of 1.5 mm as the current collector. This choice offers two primary advantages. First, titanium exhibits superior corrosion resistance to acids compared to metals such as copper. Second, the 1.5 mm diameter provides substantial mechanical robustness; its thickness makes it highly resistant to fracture, ensuring reliable operation throughout the duration of our microbial electrochemical experiments.

## Build

Following the improvement in the current collector material, we reassembled the **microbial electrochemical system** for*A. ferrooxidans* . A schematic diagram of the modified setup is presented in Figure 2-1.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f2-1.webp)

Figure 2-1 Microbial electrochemical reactor based on construction of *A. ferrooxidans*

## Test

The current-time curves were recorded, and the**current density** was calculated based on the working electrode area. The resulting current density profile over time is presented in Figure 2-2. Furthermore, to investigate the critical role of iron as an electron shuttle, we examined the system's performance with varying initial concentrations of ferrous ions, as also summarized in Figure 2-2.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f2-2.webp)

Figure 2-2 Current density-time curves of wild-type *A. ferrooxidans* (A) measured at an initial ferrous iron concentration of 159 mM (equal to the ferrous iron concentration in normal 9k-FeSO4 medium) added to the device (B) measured at an initial ferrous iron concentration of 48 mM added to the device （C）Comparison of current density-time curves

## Learn

**Having successfully established the microbial electrochemical system based on *A. ferrooxidans*,** our focus now shifts to its optimization.

Optimization of an *A. ferrooxidans*-Based Microbial Electrochemical System: Module 1 – Electron Transfer Module

## Design

**In the CUG-China 2024 project, we successfully developed a biobrick-compatible plasmid, pYDT, for *Acidithiobacillus ferrooxidans* ATCC 23270, which is expected to facilitate future research efforts by other teams. Building on this achievement, we aim to explore further synthetic biology applications of *A. ferrooxidans* in 2025.**

**During the 2024 project,**
to enhance the bioleaching efficiency of *A. ferrooxidans*, we overexpressed the diguanylate cyclase (DGC) genes to increase intracellular levels of the secondary messenger c-di-GMP, thereby promoting biofilm formation. Five DGC genes were selected for this purpose: *yedQ* (BBa\_K4242004) from *Escherichia coli*, along with native *A. ferrooxidans* genes AFE\_0053 (BBa\_K5323000), AFE\_1373 (BBa\_K5323999), and AFE\_1379 (BBa\_K5323999). Our project aimed to identify the most effective DGC gene for dynamically regulating biofilm formation in *A. ferrooxidans* by measuring both biofilm biomass and c-di-GMP concentrations, with the goal of optimizing bioleaching performance.

Subsequent transcriptomic analysis conducted by our research group revealed that engineered A. ferrooxidans strains overexpressing **DGC AFE\_0053 (BBa\_K5323000)** exhibited **significant upregulation** of **key electron transport-related genes** , including *cyc1*, *coxB*, *rus*, *coxA*, and *cyc2*.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f3-1.webp)

Figure 3-1 Results of transcriptome analysis of engineered bacteria constructed by the project in 2024 (A) Transcriptomics analysis of gene expression (B) qPCR results

Having established the *A. ferrooxidans*-based microbial electrochemical system, we are now prompted to consider how to enhance its efficiency in utilizing electrical energy for various applications, such as improved biofuel production. Given the central role of electron transfer in the metabolism of *A. ferrooxidans*, and considering that c-di-GMP serves as a key bacterial second messenger regulating diverse physiological processes—including, notably, electron transport[4]—
**we have decided to initially focus on optimizing the electron transfer efficiency in *A. ferrooxidans*, building directly upon our 2024 project findings.**

**Our prior transcriptomic data showed that overexpression of DGC AFE\_0053 upregulates electron transfer genes in *A. ferrooxidans***
, raising the question of whether this is mediated by the consequent increase in c-di-GMP. Given that c-di-GMP is known to enhance electron transfer in Geobacter,
**we will leverage our 2024 screening results and overexpress the most potent DGC, AFE\_0053, to improve electron transfer efficiency in our electrochemical system.**

## Build

The engineered *A. ferrooxidans* strains (carrying the AFE\_0053 plasmid or the empty pYDT vector) were grown in 150 mL of 9K medium (44.22 g/L FeSO₄·7H₂O) in 250 mL flasks at 30°C and 150 rpm. After centrifugation (5,500 rpm, 5 min), the cell pellets were resuspended and inoculated into the reactor at OD600 = 0.1. Streptomycin (500 μg/mL) and IPTG (1 mM) were added to the reactor medium. Following electrode connection to a potentiostat, the reactor was transferred to a 30°C incubator for electrochemical characterization.

We then quantified the**c-di-GMP levels** in the engineered strains and recorded the current-time curves. **The current density** was derived by normalizing the current to the working electrode area.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f3-2.webp)

Figure3-2 Microbial electrochemical reactor construction of engineering *A. ferrooxidans* (Plasmids containing the AFE\_0053 gene or carrying the empty plasmid pYDT ) .One of the groups (consisting of three devices) is shown here.

## Test

The resulting **current density-time profile** and the corresponding **c-di-GMP levels** are shown in Figure 3-2 and Figure 3-5, respectively.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f3-3.webp)

Figure 3-3 Current density-time curves of engineering *A. ferrooxidans* (Plasmids containing the AFE\_0053 gene or carrying the empty plasmid pYDT ) measured at an initial ferrous iron concentration of 159 mM (equal to the ferrous iron concentration in normal 9k-FeSO4 medium) added to the device

To statistically evaluate the observed differences between the p0053 and pYDT groups evident in Figure 3-2, we performed a Student's t-test using R. The results of this analysis are presented in Figure 3-3. Additionally, a corresponding p-value plot was generated and is shown in Figure 3-4.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f3-4.webp)

Figure 3-4 box plot (math.) of Current Density Comparison ( \*\*\* p < 0.001 )

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f3-5.webp)

Figure 3-5 P-value curve for each point of Current Density Comparison

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f3-6.webp)

Figure 3-6 c-di-GMP concentration of *A. ferrooxidans*. Statistical significance was determined by a two-sided Student's t-test ( \*\* 0.001 < p < 0.01).

**The p0053 engineered strain showed a significantly steeper decline in current density over time than the pYDT control, reflecting its enhanced electron utilization and implying stronger electron transfer capability under the same conditions.**

The lack of a significant difference in current density during the early phase, as supported by the p-value trajectory, is attributable to initial bacterial growth and biofilm development, where variations in electron transfer were not yet pronounced.

**As the biofilm matured, the higher c-di-GMP level in the p0053 strain facilitated markedly stronger electron transfer, resulting in a rapid drop in current density.**
In contrast, the pYDT group decayed more gradually due to its weaker electron transfer capacity.
**These findings indicate that matured biofilm magnifies inherent differences in electron transfer efficiency, with the p0053 strain demonstrating superior performance in the later phase.**

## Learn

Compared to the control group, the p0053 engineered strain demonstrated a stronger electron transfer capacity.
**The successful operation of this high-efficiency microbial electrochemical system highlights the potential of *A. ferrooxidans* for applications in electrical energy storage.**

Module 2 – Glycerol Production Module

## Design

**The rapid development of renewable energy sources, particularly solar and wind power, faces a critical bottleneck in grid integration: the challenge of electricity storage.**
These energy sources are characterized by significant intermittency and volatility, with their peak generation periods often misaligned with societal electricity demand peaks, leading to substantial curtailment ("wind and solar abandonment"). To achieve a stable energy supply, the grid urgently requires a time-shifting "battery" solution that can store surplus electricity during periods of abundance and release it when needed.

However, traditional storage methods such as pumped hydro are constrained by geographical requirements, while emerging storage technologies have yet to fully meet the demands for grid-scale, long-duration energy storage in terms of scalability, cost-effectiveness, and storage duration[5].

**To address this challenge, converting electrical energy into chemical energy for storage has emerged as one of the most promising pathways.**
Beyond conventional electrochemical storage systems represented by lithium-ion batteries,
**a more cutting-edge approach involves microbial electrochemical synthesis systems. These systems utilize specific microorganisms as "biocatalysts" to directly convert simple inorganic compounds, such as carbon dioxide and water, into energy-rich green fuels (e.g., methane, formate) or high-value chemicals, driven by electrical energy[6].**

**This process effectively transforms fluctuating electricity into chemical energy carriers that are suitable for long-term storage and transportation.**
It not only offers a revolutionary new strategy for large-scale, long-duration energy storage but also enables the circular utilization of carbon resources, paving a novel technological path toward establishing a carbon-neutral energy system.

In industrial applications, glycerol—often referred to as the "industrial monosodium glutamate"—serves as an essential chemical feedstock widely used across multiple sectors. Its core value lies in its excellent hygroscopicity, moisturizing capacity, non-toxicity, and mild properties, making it a key humectant in cosmetics and toothpaste to enhance product moisturizing effects. In the food industry, glycerol acts as both a sweetener and humectant, helping to maintain the soft texture of confectionery, baked goods, and other food products. Furthermore, glycerol is extensively used in the manufacture of explosives (e.g., nitroglycerin), synthetic resins, lubricants, and antifreeze agents[7].

**Given glycerol’s significant industrial utility and the relative maturity of microbial metabolic engineering for its large-scale production, this project proposes the introduction of an exogenous glycerol synthesis pathway to redirect carbon flux toward glycerol production.**

**To further enhance glycerol synthesis efficiency, we introduced the glycerol biosynthesis pathway from *Saccharomyces cerevisiae*,**
which relies on the key enzymes encoded by *gpd1* and *gpp2*. To optimize the catalytic efficiency of these enzymes,
**a fusion protein was designed by linking *gpd1* and *gpp2* via a flexible linker sequence**
, enabling spatial organization that facilitates more efficient glycerol production. In addition,
**to improve glycerol extraction, we incorporated the gene *fps1*, which encodes a glycerol channel protein (aquaglyceroporin).**
This enables efficient transport of intracellular glycerol to the extracellular environment, thereby streamlining downstream recovery and separation processes.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/engineeringnew01.webp)

Figure 4-1 Gene circuit of the Glycerol Synthesis and Exocytosis Module

We performed **preliminary codon optimization** of the sequences for *A. ferrooxidans* and designed the linker sequence between *gpd1* and *gpp2*. The codon-optimized gene encoding the Gpd1–Gpp2 fusion protein, as well as the *fps1* gene, were synthesized by Tsingke Biotechnology Co., Ltd. These genes were subsequently cloned into the plasmid pYDT, a step also carried out by Tsingke Biotechnology Co., Ltd.

## Build

Upon obtaining the expression vector pFps1-fus(pYDT-Ptac-fps1-gpd1-gpp2fus) and the empty vector pYDT, they were transformed into *E. coli* DH5α competent cells. Colony PCR was performed to screen for clones successfully transformed with the target plasmids. The plasmids were then extracted and analyzed by agarose gel electrophoresis. Those showing the expected band size were subjected to sequencing to confirm sequence accuracy. Once correct sequences were verified, 700 µL of bacterial culture was mixed with an equal volume of glycerol (1:1 ratio), and the mixtures were stored at –80°C for long-term strain preservation.

**We plan to validate the function of the glycerol production module by expressing the vectors in the prokaryotic model strain *E. coli* BL21. Both pFps1-fus and the empty vector pYDT will be introduced into *E. coli* BL21. Colony PCR will again be used to identify correctly transformed clones. Glycerol levels in the fermentation broth will be measured to evaluate the production efficiency of the engineered *E. coli* BL21 strains carrying the target module.**
The correct expression vectors will be transformed into the **conjugation** donor strain *E. coli* SM10 for subsequent conjugation with the recipient bacterium *A. ferrooxidans*. Colony PCR will be used to screen and select *A. ferrooxidans* clones that have been successfully transformed with the target plasmids, which will then be subjected to larger-scale cultivation.

Following genomic DNA extraction from the selected *A. ferrooxidans* clones, PCR amplification of the 16S rDNA sequence will be performed. The amplified sequences will be compared to confirm the identity of the engineered *A. ferrooxidans*. In the verified strains, glycerol levels in the fermentation broth will be quantitatively analyzed.

The glycerol assay is based on a coupled chemical reaction sequence involving the Malaprade reaction and the Hantzsch reaction. The reactions are carried out in a 96-well plate, and kinetic measurements are monitored using a microplate reader. Absorbance at 410 nm is recorded over a period of 25 minutes. Glycerol concentration is calculated according to a glycerol standard curve using Equation 1 (detailed principles and procedures for glycerol measurement are available on the protocol).

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/notebook/notebooke.avif)

## Test

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f4-2.webp)

Figure 4-2 PCR results of *E. coli* BL21 colonies

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/engineering/engineeringnew.webp)

Figure 4-3 Growth curves of the engineered strain under varying concentrations of IPTG inducer. The IPTG concentrations in each panel are as follows: (A) 0 mM, (B) 0.1 mM, (C) 0.5 mM, and (D) 1 mM. All experiments were conducted in triplicate, and error bars indicate the standard deviation (SD).

To determine whether the glycerol production module could successfully synthesize and export glycerol, we initially conducted a study using *E. coli* BL21 as the model strain. The bacteria were cultured in
**M9 medium with different IPTG concentrations (0 mM, 0.1 mM, 0.5 mM, and 1 mM), and growth curves**
were plotted. As shown in Figure 4-3, the absence of IPTG (0 mM) had no effect on the growth of either strain, providing a baseline control for the other experimental groups.

Across the various IPTG concentrations, the strain carrying the empty vector pYDT generally exhibited better growth than the pFps1-fus strain, indicating that IPTG exerts an inhibitory effect on the growth of the engineered bacteria, with the inhibition becoming more pronounced as the concentration increased. Based on a comprehensive comparison,
**an IPTG concentration of 0.5 mM was identified as optimal, balancing efficient target gene expression with acceptable bacterial growth.**

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f4-4.webp)

Figure 4-4 Glycerol standard curve. The R-squared value of 0.9914 demonstrates excellent linearity of the calibration.

![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f4-5-1.webp)
![Figure 1. Annual mean globally averaged atmospheric mole fraction of carbon dioxide from 1984 to 2023 in parts per million (ppm) Source: Data are from the World Data Centre for Greenhouse Gases (WDCGG)](https://static.igem.wiki/teams/5665/engineering/f4-5-2.webp)

Figure 4-5 Glycerol production under varying IPTG induction. (A, B) Glycerol titers at 11 and 23 hours, respectively, across a range of IPTG concentrations. (C, D) Comparison of glycerol production between 11 and 23 hours for E. coil harboring the pYDT plasmid (C) or expressing the Fps1 transporter (D). Data are presented as the mean ± SD from three independent replicates (n=3). Statistical significance was determined by a two-sided Student's t-test ( \* p < 0.05).

Based on the glycerol standard curve shown in Figure 4-4, we calculated the glycerol production levels at different IPTG concentrations. The results indicate that the highest glycerol yield was achieved at an IPTG concentration of 0.5 mM, with a statistically significant increase compared to the empty vector control.
**The maximum glycerol accumulation was observed during the late logarithmic growth phase.**
As shown in Figure 4-5 A and Figure 4-5 B, glycerol production in the experimental group (pFps1-fus) consistently exceeded that of the empty vector group.
**Specifically, at 0.5 mM IPTG, the engineered strain produced significantly more glycerol than the control.**
This can be attributed to the higher metabolic burden imposed by increased IPTG concentrations, which diverts cellular energy toward maintenance and recombinant protein synthesis rather than biomass formation.

Further comparison of glycerol yields at different time points (Figure 4-5 C and Figure 4-5 D) reveals that production at 11 hours was higher than at 23 hours. This pattern can be explained by the growth phase of the culture: around 11 hours, corresponding to the late logarithmic phase, the bacterial population had largely completed active growth and consumed most of the available nutrients in the medium. As the culture entered the stationary phase, the remaining nutrients were likely sufficient only to support basic cellular functions, leaving limited resources for additional glycerol synthesis.

## Learn

Growth curve data indicate that the engineered strain pFps1-fus consistently exhibited slower growth than the empty vector control (pYDT), which can be attributed to its higher metabolic burden. This growth retardation was further exacerbated at elevated concentrations of the inducer IPTG.
 **Such slow growth inevitably leads to extended cultivation times. Therefore, we aim to develop strategies to accelerate the growth of *A. ferrooxidans* in subsequent phases of this project.**

Domestication of *A. ferrooxidans*: From Autotrophy to Heterotrophy

## Design

There exists a significant difference in growth and proliferation rates between heterotrophic and autotrophic microorganisms, with the former typically growing much faster than the latter. This divergence stems from their fundamentally distinct strategies for acquiring energy and carbon sources. Heterotrophs, such as *Escherichia coli*, can directly utilize readily available organic compounds from the environment, enabling efficient and rapid energy acquisition. Under favorable conditions, this allows them to achieve remarkably short generation times—as brief as 20 minutes.

In contrast, autotrophic microorganisms must rely on light energy or the oxidation of inorganic substances to fix carbon dioxide and synthesize all organic materials de novo. This process is not only metabolically complex but also characterized by low energy conversion efficiency, resulting in considerably slower growth and metabolism. Consequently, their generation times often extend to several hours or more.

Thus, the fundamental difference in their "foraging" strategies underlies the vast disparity in growth rates observed between these two groups of microorganisms[8].

**As a typical chemolithoautotrophic bacterium, *A. ferrooxidans* exhibits an extremely slow growth rate, which poses significant challenges for laboratory research.** Its prolonged cultivation cycle considerably delays experimental progress and results in low data output efficiency.
However, the boundaries of metabolic types are not rigid, leading to the concept of "domesticating" and engineering microbial metabolic capabilities. Interestingly, even obligate chemolithoautotrophs may possess metabolic blueprints that retain hidden "shortcuts" similar to those of heterotrophic organisms.
**A notable example is *A. ferrooxidans*: while it primarily obtains energy from the oxidation of ferrous iron or reduced sulfur compounds and fixes CO₂ as its main carbon source, it also retains complete glycolysis and tricarboxylic acid (TCA) cycle pathways. This metabolic capacity suggests the potential to guide the bacterium toward mixotrophic or even heterotrophic growth through gradual exposure to organic carbon sources, thereby overcoming its inherent growth limitations.**

**To test this hypothesis, we designed a series of media for stepwise domestication of *A. ferrooxidans* from autotrophy toward heterotrophy, as summarized in Table 5-1.**The standard 9K-FeSO4 medium contains 44.22 g/L ferrous sulfate. In our adaptation protocol, the glucose concentration was progressively increased, while ferrous iron was systematically reduced to 100%, 50%, 20%, and finally 0% of its original level. The specific concentrations of glucose and ferrous iron in each medium formulation are provided in Table 5-1.

Table 5-1 Domestication medium of *A. ferrooxidans*

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/table5.webp)

## Build

The 9K-FeSO4 media were prepared according to the formulations specified in Table 5-1 (detailed preparation procedures are available on the notebook page). Wild-type *A. ferrooxidans* was first inoculated into Acclimation 1 medium for expansion culture. The resulting culture was then transferred sequentially into Acclimation 2 medium, followed by Acclimation 3 and finally Acclimation 4 medium. Throughout this domestication process, the growth curve of *A. ferrooxidans* and the concentration changes of ferrous iron in each medium were monitored.

## Test

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f-5-1-final.webp)

Figure 5-1 Growth curves of domesticated *A. ferrooxidans*

**A previous study performing full-wavelength scanning on *A. ferrooxidans* cultures in 9K-FeSO₄ medium identified the highest absorbance peak at λmax = 420 nm.**
Consequently, for cultures grown on ferrous iron, it is recommended to measure cell density at OD₄₂₀ rather than OD₆₀₀, as the latter yields relatively low and less reliable readings under certain circumstances[9].

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f-5-2-final.webp)

Figure 5-2 Changes in the concentration of ferrous iron in the medium

**No bacterial growth was observed in the Acclimation 4 group**
, and only minor fluctuations in ferrous iron concentration were detected. These results indicate that when the domestication process reached the Acclimation 4 stage,
**the strain failed to establish an efficient heterotrophic metabolic system utilizing glucose as the primary carbon source,**
while its innate iron-oxidizing lithoautotrophic mode was also compromised due to the progressive adaptation pressure, ultimately leading to growth arrest.

This outcome reveals that the metabolic adaptability of *A. ferrooxidans*—from iron-based autotrophy to glucose-based heterotrophy—has a distinct physiological limit. The conditions in Acclimation 4 appear to exceed the metabolic plasticity of the strain, surpassing its capacity for physiological adjustment.

## Learn

Electron transfer plays a critical role in the physiology of *A. ferrooxidans*.
**Its chemolithoautotrophic metabolism, which relies on the oxidation of ferrous iron or reduced sulfur compounds, is a deeply ingrained trait shaped by long-term evolution.**
This inherent nutritional mode is difficult to fundamentally alter through short-term artificial domestication. Achieving fully heterotrophic growth in A. ferrooxidans may require an impractically long experimental timeline and numerous domestication steps—contradicting our original goal of simply accelerating its growth rate to shorten research cycles.
Therefore, it may be prudent to
**shift our strategy and explore alternative metabolic pathways in *A. ferrooxidan***that could be modulated to enhance growth without requiring a full trophic transition.

Module 3 – CO2 Fixation Module

## Design

Recognizing the challenges in steering the organism toward organoheterotrophy through domestication, we have shifted our research focus to a more fundamental strategy:
**enhancing the intrinsic "productivity" of *A. ferrooxidans* by strengthening its core carbon fixation capacity, thereby addressing the growth bottleneck from within.**
This approach aims to empower its native autotrophic growth rather than altering its trophic mode.

Biochemical reactions within the cell are primarily catalyzed by enzymes, among which key enzymes—also known as rate-limiting enzymes—play a decisive regulatory role in metabolic pathways[10].
**In *A. ferrooxidans*, carbon dioxide fixation mainly depends on the Calvin–Benson–Bassham (CBB) cycle**
[11], a process governed by two key enzymes:
**phosphoribulokinase (PRK)** and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO).

We selected PRK enzymes from ten different organisms, as listed in Table 6-1. Their structural files were either directly obtained from the**UniProt** database or predicted using **AlphaFold**. Molecular docking with the substrate Ru5P was performed using **AutoDock Vina**, and two PRK variants were selected based on their
**binding energies. In addition, we included the native PRK enzyme from *A. ferrooxidans* itself—a decision informed by our prior experience from the 2024 project.**

Table 6-1 List of organisms from which PRK gene originates

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/table6.webp)

We experimentally validated the activity of the three selected PRK enzymes. To enable appropriate expression in *A. ferrooxidans*, we optimized the codon usage of the PRK genes. We hypothesize that overexpressing PRK will enhance the efficiency of carbon dioxide fixation, accelerate the accumulation of organic carbon compounds, and ultimately increase the bacterial growth rate.

Based on the native PRK sequence from the *A. ferrooxidans* genome, we designed primers and added restriction enzyme sites to both ends of the gene via PCR amplification.
**Using a double-digestion method, both the target gene and the vector were digested to generate compatible sticky ends, followed by ligation with T4 DNA ligase to construct the expression vector.**

For the heterologous PRK genes (RS0121050 and RS05100), we performed preliminary codon optimization for *A. ferrooxidans*. The codon-optimized RS0121050 and RS05100 genes were synthesized by Tsingke Biotechnology Co., Ltd. and subsequently cloned into the plasmid pYDT, a step also completed by Tsingke.

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f6-1.webp)

Figure 6-1 Construction of PRK expression vectors by double digest method

## Build

**Since the CDS of AFE\_0536 contains restriction sites used in our BioBrick assembly system, we decided to employ overlap extension PCR to introduce specific mutations into this sequence.**

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f6-2.webp)

Figure 6-2 Schematic of the use of overlapping extension PCR for targeted mutagenesis

**During the third round of PCR amplification, we simultaneously amplified the wild-type AFE\_0536 gene as a control for the site-directed mutagenesis. The resulting products were then verified using a single-enzyme digestion assay.**
Upon obtaining the expression vectors pAFE\_0536, pRS0121050, pRS05100, and the empty vector pYDT, they were transformed into *E. coli* DH5α competent cells. Colony PCR was used to screen for clones successfully transformed with the correct plasmids. Positive clones were subjected to plasmid extraction and analyzed by agarose gel electrophoresis. Plasmids displaying the expected fragment sizes were sequenced to confirm sequence accuracy. Once correct sequences were verified, 700 μL of bacterial culture was mixed with an equal volume of glycerol (1:1 ratio), and the aliquots were stored at –80°C for long-term strain preservation.
We plan to **evaluate the effect of PRK overexpression**by introducing the expression vectors into **the prokaryotic model strain *E. coli* BL21**The vectors pAFE\_0536, pRS0121050, pRS05100, and the empty control pYDT will be transformed into *E. coli* BL21. Colony PCR will be used to screen for correctly transformed clones. Growth curves will be measured for the verified transformants to assess physiological impacts.

Subsequently, the confirmed expression vectors will be transformed into the conjugation donor strain *E. coli* SM10 for intergeneric conjugation with the recipient *A. ferrooxidans*. Colony PCR will again be employed to screen for *A. ferrooxidans* transconjugants carrying the target plasmids, which will then be expanded in larger-scale cultures.

Genomic DNA will be extracted from the putative *A. ferrooxidans* transconjugants and subjected to 16S rDNA amplification and sequencing to confirm species identity. In the verified engineered strains, growth curves and temporal changes in ferrous iron concentration in the medium will be monitored.

## Test

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f6-3.webp)

Figure 6-3 Validation of targeted mutation results by overlapping extension PCR

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f6-4.webp)

Figure 6-4 PCR results of *E.coli* BL21 colonies

**We successfully performed site-directed mutagenesis on AFE\_0536 using overlap extension PCR and obtained the modified sequence. The expression vectors pRS0121050 and pRS05100, together with the empty vector pYDT, were successfully introduced into *E. coli* BL21.**

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f6-5-1.webp)
![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f6-5-2.webp)

Figure 6-5. The impact of IPTG induction on the growth of *E. coli* BL21. (A-D) Growth curves monitored in LB medium supplemented with 0, 0.1, 0.5, and 1 mM IPTG. (E) Comparison of growth profiles for the pYDT strain across the different IPTG concentrations. (F) Comparison of growth profiles for the pRS0121050 strain across the different IPTG concentrations. All experiments were performed in triplicate, and error bars represent the standard deviation (SD).

As shown in Figure 6-5, the growth curves of the pYDT strain showed no significant differences across various IPTG concentrations, indicating that IPTG itself has no substantial impact on the growth or physiological state of the empty vector control. In contrast, the pRS0121050 strain exhibited optimal growth in the absence of IPTG (0 mM), with a gradual decline in growth capacity as IPTG concentrations increased.

**These results suggest that higher IPTG concentrations lead to elevated expression of phosphoribulokinase (PRK), and that high-level PRK expression imposes a metabolic burden on the cells, thereby inhibiting bacterial growth.**
As presented in Figure 6-6, growth curves of *E. coli* BL21 carrying pYDT and pRS05100 under different IPTG concentrations consistently showed that pRS05100 grew more slowly than the empty vector control.
**Furthermore, as the IPTG concentration increased, the growth inhibitory effect was enhanced in a dose-dependent manner, with higher induction levels resulting in progressively slower growth rates.**

![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f6-6-1.webp)
![Figure 3. Schematic diagram of the principle of solar panels](https://static.igem.wiki/teams/5665/engineering/f6-6-2.webp)

Figure 6-6 Growth curves of *E. coli* BL21 harboring pYDT (*E. coli* BL21/pYDT) and pRS0121050 (*E. coli* BL21/ pRS0121050) in LB medium under induction with varying concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG) are presented as follows: (A) 0 mM IPTG, (B) 0.1 mM IPTG, (C) 0.5 mM IPTG, and (D) 1 mM IPTG. Additionally, growth curves for strains (E) *E. coli* BL21/pYDT and (F) *E. coli* BL21/ pRS0121050 are compared across the different IPTG concentrations. The data are expressed as the mean of three independent replicates (n=3), with error bars indicating standard deviation (SD).

Based on the collective results,
**the PRK enzyme expressed from the recombinant vector pYDT\_RS0121050 appears to be more effective than that from**

## Learn

We have now established a complete project workflow for this study. By seamlessly integrating the three modules described above,
**our team is committed to developing a sustainable and efficient approach for electrical energy storage and carbon fixation using a microbial electrosynthesis system based on *A. ferrooxidans*.**
This innovative strategy not only aims to construct a functional *A. ferrooxidans*-driven electrochemical platform but also offers a promising solution to address energy and climate challenges, aligning with the growing demand for environmentally friendly technologies.
**Our work establishes a foundational framework for this field, having successfully implemented a prototype microbial electrosynthesis system using *A. ferrooxidans*. This opens new avenues for advancing the role of *A. ferrooxidans* in microbial electrochemistry. We hope that more research teams will engage in exploring *A. ferrooxidans*, expanding its application potential in microbial electrosynthesis and related areas, thereby fully realizing its considerable research value.**

References

* [1] Kernan, T., Majumdar, S., Li, X., Guan, J., West, A. C., & Banta, S. (2016). Engineering the iron-oxidizing chemolithoautotroph *Acidithiobacillus ferrooxidans* for biochemical production. *Biotechnology and Bioengineering, 113*(1), 189–197.
* [2] Rabaey, K., & Rozendal, R. A. (2010). Microbial electrosynthesis: Revisiting the electrical route for microbial production. *Nature Reviews Microbiology, 8*(10), 706–716.
* [3] Sathiyanarayanan, G., Chabert, N., Tulumello, J., & Achouak, W. (2021). Building efficient biocathodes with *Acidithiobacillus ferrooxidans* for the high current generation. *Journal of Power Sources, 514*, 230586.
* [4] Hu, Y., Han, X., Luo, Y., Wang, Y., Li, C., Zhang, T., & Liu, Y. (2024). All roads lead to Rome: Cyclic di-GMP differentially regulates extracellular electron transfer in Geobacter biofilms. *The Innovation Life, 2*(1), 100052.
* [5] Conrado, R. J., Haynes, C. A., Haendler, B. E., & Toone, E. J. (2013). Biofuel development: The role of catalytic conversion. In J. W. Lee (Ed.), *Advanced biofuels and bioproducts* (pp. 1037–1064). Springer.
* [6] Berg, J. M., Tymoczko, J. L., Gatto, G. J., & Stryer, L. (2015). Biochemistry (8th ed.). W.H. Freeman.
* [7] Tan, H. W., Aziz, A. A., & Aroua, M. K. (2013). Glycerol production and its applications as a raw material: A review. *Renewable and Sustainable Energy Reviews, 27*, 118–127.
* [8] Sanyal, A., Antony, R., Samui, G., & Subudhi, S. (2024). Autotrophy to heterotrophy: Shift in bacterial functions during the melt season in Antarctic cryoconite holes. *Journal of Microbiology, 62*(8), 591–609.
* [9] Wang, Q., Long, H., Wang, H., & Lau Vetter, M. C. Y. (2024). Characterize the growth and metabolism of *Acidithiobacillus ferrooxidans* under electroautotrophic and chemoautotrophic conditions. *Microorganisms, 12*(3), 590.
* [10] Nelson, D. L., & Cox, M. M. (2021). *Lehninger principles of biochemistry* (8th ed.). W.H. Freeman.
* [11] Valdés, J., Pedroso, I., Quatrini, R., Dodson, R. J., Tettelin, H., Blake, R., Eisen, J. A., & Holmes, D. S. (2008). *Acidithiobacillus ferrooxidans* metabolism: From genome sequence to industrial applications. *BMC Genomics, 9*, Article 597.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
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## What Should this Page Contain?

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Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block extra\_css %}
{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5665/first-photo/f-project.webp)

Results

Result one

Result two

Result three

References

I. Successful Construction of an *Acidithiobacillus ferrooxidans*-Based Microbial Electrochemical System with an Efficient Electron Transfer Module

In the 2024 project, we constructed engineered *A. ferrooxidans* strains with elevated levels of c-di-GMP by overexpressing c-di-GMP synthases (Figure 1-1). In subsequent studies conducted by our research group on the engineered bacterial strains constructed during the 2024 project, transcriptomic analysis revealed that the overexpression of DGC AFE\_0053 (BBa\_K5323000) led to significant up regulation of electron transport-related genes in *A. ferrooxidans* (Figure 1-2).

![Figure 1-1 c-di-GMP Content in Engineered Strains Constructed in the 2024 Project. Statistical significance was determined by a two-sided Student's t-test ( * 0.01 < p < 0.05, ** 0.001 < p < 0.01 , *** p < 0.001).](https://static.igem.wiki/teams/5665/results/r1-1.webp)

*A. ferrooxidans* thrives in highly acidic environments (pH 1.5–2.5) by oxidizing ferrous iron (Fe2+) to obtain energy, a process in which electron transfer plays a central role. Iron, being a multivalent metal, can exist in solution as either Fe2+ or Fe3+, making it an effective electron shuttle. It has been reported that *A. ferrooxidans* utilizes soluble iron species as electron shuttles[1].

Based on these findings, we propose to construct a microbial electrochemical system using *A. ferrooxidans* as the chassis organism to explore its electrochemical applications.

Current-time curves were recorded, and the current density was calculated by normalizing the current to the working electrode area (1 cm × 3 cm=3 cm2). The resulting current density-time profile is presented in Figure 1-3. To further investigate the role of iron as an electron shuttle, we also examined the system's behavior under varying initial concentrations of ferrous ions, as summarized in the same figure.

![Figure 1-1 c-di-GMP Content in Engineered Strains Constructed in the 2024 Project. Statistical significance was determined by a two-sided Student's t-test ( * 0.01 < p < 0.05, ** 0.001 < p < 0.01 , *** p < 0.001).](https://static.igem.wiki/teams/5665/results/r1-3.webp)

Figure 1-3 Current density-time curves of wild-type *A. ferrooxidans* (A) measured at an initial ferrous iron concentration of 159 mM (equal to the ferrous iron concentration in normal 9k-FeSO4 medium) added to the device (B) measured at an initial ferrous iron concentration of 48 mM added to the device (C) Comparison of current density-time curves.

We measured the intracellular c-di-GMP levels in the electron transfer module engineered strains and recorded the current-time curves. The current density was calculated based on the working electrode area. The current density-time profile is presented in Figure 1-4.

![Figure 1-1 c-di-GMP Content in Engineered Strains Constructed in the 2024 Project. Statistical significance was determined by a two-sided Student's t-test ( * 0.01 < p < 0.05, ** 0.001 < p < 0.01 , *** p < 0.001).](https://static.igem.wiki/teams/5665/results/r1-4.webp)

Figure 1-4 Current density-time curves of engineering *A. ferrooxidans* strains harbouring plasmids with the AFE\_0053 gene or carrying the empty plasmid pYDT measured at an initial ferrous iron concentration of 159 mM (equal to the ferrous iron concentration in normal 9k-FeSO4 medium) added to the device.

Figure 1-4 illustrates the current density trends of the engineered p0053 group and the pYDT group over time. Overall, both groups exhibited a declining trend in current density (toward more negative values), but the decrease was markedly more pronounced in the p0053 group. By approximately 300 hours, the current density of the p0053 group approached -20 A/m², whereas the pYDT group remained above -15 A/m². These results indicate that, over extended operation, the p0053 strain demonstrates a significantly faster and greater decline in current density compared to the control, reflecting its enhanced electron utilization capability. Collectively, under identical conditions, the p0053 engineered strain likely possesses stronger electron transfer capacity.

![Figure 1-1 c-di-GMP Content in Engineered Strains Constructed in the 2024 Project. Statistical significance was determined by a two-sided Student's t-test ( * 0.01 < p < 0.05, ** 0.001 < p < 0.01 , *** p < 0.001).](https://static.igem.wiki/teams/5665/results/r1-5.webp)

Figure 1-5 (A) box plot (math.) of Current Density Comparison. (\*\*\* p < 0.001) (B) P-value curve for each point of Current Density Comparison

We further performed a Student's t-test using R to statistically evaluate these differences. The results of the t-test are presented in Figure 1-5 (A). Additionally, a corresponding p-value plot was generated and is shown in Figure 1-5 (B). As shown in Figure 1-5(A), a statistically significant difference in current density was observed between the engineered p0053 group and the pYDT control group (denoted by, \*\*\* p < 0.001). Specifically, the p0053 group exhibited an average current density of -7.53 ± 7.01 A/m², compared to -5.40 ± 4.50 A/m² for the pYDT group. The lower (more negative) mean current density, along with greater variability (standard deviation) in the p0053 group, indicates not only a significant divergence in current output between the two groups but also higher data dispersion within the engineered strain.
Figure 1-5(B) displays the p-values from statistical significance analysis of current density differences between the engineered p0053 and pYDT groups over time, presented as -log10(p-value). During the initial phase of the experiment (approximately 0–250 hours), the -log10(p-value) remained close to zero, indicating no significant difference between the two groups. However, after approximately 250 hours, the p-value decreased sharply (reflected by a sharp increase in -log10(p-value)), reaching an extremely high level of statistical significance ( \*\*\* p < 0.001). This trend aligns with the patterns observed in the previous two figures, further confirming that significant differences in current density between the groups emerged and became statistically robust during the later stage of the experiment.

During the initial phase of the experiment, no significant difference in current density was observed between the engineered p0053 group and the pYDT control group, as indicated by the corresponding p-value curve showing non-significance in this period. This phenomenon can be largely attributed to the stages of bacterial proliferation and initial biofilm formation, during which differences in electron transfer capacity had not yet been fully manifested.

Over time, as the biofilm matured and bacterial density reached a stable level, the p0053 group—benefiting from higher intracellular c-di-GMP concentrations—exhibited enhanced electron transfer capability, leading to a rapid decline (more negative shift) in current density. In contrast, the pYDT group, with weaker electron transfer capacity, showed a more gradual decay. Thus, upon biofilm maturation, the disparity in electron transfer efficiency became the dominant factor governing current density dynamics, with the p0053 strain demonstrating significantly higher electron transfer efficiency in the later stage, resulting in a pronounced difference in current density compared to the control.

In summary, we have successfully established a microbial electrochemical system based on *A. ferrooxidans* and constructed an efficient electron transfer module.

II. Successful Construction of a Module for Glycerol Synthesis and transport

To promote efficient glycerol synthesis in engineered bacteria and enable successful glycerol efflux, we selected and introduced three gene fragments into the engineered bacteria, namely Fission yeast polyol transporter 1 (Fps1), Glycerol-3-phosphate dehydrogenase (NAD(+)) (Gpd1), and glycerol-1-phosphatase (Gpp2), which are derived from *Saccharomyces cerevisiae* S288C. A sequence encoding the GGGGS amino acid peptide was inserted between Gpd1 and Gpp2, forming a Gpd1-Gpp2 fusion protein to facilitate intracellular glycerol synthesis in the engineered bacteria. *fps1* encodes a glycerol channel protein, which enables glycerol efflux. During the engineering phase, we constructed a recombinant plasmid pFps1-fus. This plasmid uses the IPTG-inducible Ptac promoter to regulate the expression of the Fps1-fus fusion protein.

To evaluate whether these genes can successfully synthesize glycerol and enable glycerol efflux, we first conducted studies using *E. coli* BL21 as the model strain. We set up different IPTG concentrations (0 mM, 0.1 mM, 0.5 mM, and 1 mM), cultured the strain in M9 medium, and plotted the growth curve.

As shown in Figure 2-1, when IPTG concentration was 0 mM, there was no effect on the growth of either strain, providing a control for other experimental groups. At different IPTG concentrations, the growth of the pYDT strain was generally better than that of the pFps1-fus strain. This indicates that IPTG inhibits the growth of the engineered bacteria, and the inhibitory effect becomes more pronounced as the IPTG concentration increases. Comprehensive comparison showed that when IPTG concentration was 0.5 mM, it was not only beneficial for the expression of the target genes but also favorable for the growth of the engineered bacteria.

![Figure 2. A high-level overview of the concepts associated with bioelectrochemical systems.](https://static.igem.wiki/teams/5665/results/r2-1.webp)

Figure 2-1 Growth curves of the engineered strain under varying concentrations of IPTG inducer. The IPTG concentrations in each panel are as follows: (A) 0 mM, (B) 0.1 mM, (C) 0.5 mM, and (D) 1 mM. All experiments were conducted in triplicate, and error bars indicate the standard deviation (SD).

To further investigate glycerol production capacity and the impact of IPTG, thereby identifying the optimal induction level, we quantified glycerol yields in both strains[2]. The corresponding glycerol standard curve is shown in Figure 2-2.

![Figure 2. A high-level overview of the concepts associated with bioelectrochemical systems.](https://static.igem.wiki/teams/5665/results/r2-2.webp)

Figure 2-2 Glycerol standard curve. The R-squared value of 0.9914 demonstrates excellent linearity of the calibration.

Glycerol production under different IPTG concentrations was calculated based on the standard curve. Experimental results demonstrated that the highest glycerol yield was achieved in the engineered strain at 0.5 mM IPTG, showing a statistically significant increase over the empty vector control. Peak glycerol accumulation consistently occurred during the late logarithmic phase.

As shown in Figures 2-3A and 2-3B, glycerol production in the experimental group exceeded that of the empty vector group across conditions. Specifically, at an IPTG concentration of 0.5 mM, the engineered strain produced significantly more glycerol. This can be attributed to the substantial metabolic burden imposed by higher IPTG concentrations, which forces cells to divert more energy towards maintenance rather than product synthesis.

Furthermore, comparison of glycerol yields at different time points (Figures 2-3C and 2-3D) revealed higher production at 11 hours compared to 23 hours. This pattern is explained by the fact that around 11 hours—corresponding to the late logarithmic phase—bacterial colonies had largely completed active growth and had extensively consumed available nutrients. Subsequently, growth rates declined, and the remaining nutrients in the culture were likely only sufficient to support basic cellular activities, leaving no surplus for additional glycerol synthesis.

![Figure 2. A high-level overview of the concepts associated with bioelectrochemical systems.](https://static.igem.wiki/teams/5665/results/r2-3-1.webp)
![Figure 2. A high-level overview of the concepts associated with bioelectrochemical systems.](https://static.igem.wiki/teams/5665/results/r2-3-2.webp)

Figure 2-3 Glycerol production under varying IPTG induction. (A, B) Glycerol titers at 11 and 23 hours, respectively, across a range of IPTG concentrations. (C, D) Comparison of glycerol production between 11 and 23 hours for *E. coil* BL21 harboring the pYDT plasmid (C) or expressing the Fps1 transporter (D). Data are presented as the mean ± SD from three independent replicates (n=3). Statistical significance was determined by a two-sided Student's t-test ( \* p < 0.05).

III. Successful Construction of a High-Efficiency Carbon Fixation Module for the CBB Cycle

The Calvin-Benson-Bassham (CBB) cycle is the primary carbon fixation pathway in *A. ferrooxidans*. Among the enzymes involved, phosphoribulokinase (PRK) is a key enzyme of the CBB cycle. Therefore, overexpressing PRK can improve the efficiency of the CBB cycle, thereby enhancing the carbon fixation capacity of *A. ferrooxidans*.
To achieve this goal, we aimed to screen the optimal gene fragment between PCC6301pg\_RS05100 and ACIR131C\_RS0121050. Accordingly, we constructed recombinant vectors pYDT\_RS05100 and pYDT\_RS0121050, respectively. Both vectors use an IPTG-inducible promoter to regulate the expression of the PRK-encoding gene sequence derived from *E. coli* BL21.

To evaluate which of pYDT\_RS05100 or pYDT\_RS0121050 exhibits higher PRK activity—thereby accelerating the CBB cycle and improving carbon fixation efficiency in *A. ferrooxidans*—we initially used *E. coli* BL21 as the model organism. First, we investigated suitable culture conditions and assessed the growth curves in LB medium supplemented with IPTG at induction concentrations of 0 mM, 0.1 mM, 0.5 mM, and 1 mM, respectively.

As shown in Figure 3-1, under different IPTG concentrations, the growth curves of the pYDT strain showed no significant differences. This indicates that IPTG itself has no obvious effect on the growth or physiological state of the empty vector strain (i.e., the pYDT strain). In contrast, the pRS0121050 strain exhibited the best growth when the IPTG concentration was 0 mM, and its growth capacity gradually decreased as the IPTG concentration increased. This result demonstrates that higher IPTG concentrations lead to increased expression levels of phosphoribulokinase (PRK); however, the high expression of PRK imposes a metabolic burden on the cells, thereby inhibiting bacterial growth.

As shown in Figure 3-2, even under different IPTG concentrations, the growth curves of *E. coli* BL21 harboring pYDT and pRS05100 both indicated that the growth rate of pRS05100 was slower than that of pYDT. Furthermore, with the increase in IPTG concentration, the growth inhibitory effect was further exacerbated in a dose-dependent manner, with higher induction levels resulting in slower growth rates.

![Figure 2. A high-level overview of the concepts associated with bioelectrochemical systems.](https://static.igem.wiki/teams/5665/results/r3-1-1.webp)
![Figure 2. A high-level overview of the concepts associated with bioelectrochemical systems.](https://static.igem.wiki/teams/5665/results/r3-1-2.webp)

Figure 3-1 The impact of IPTG induction on the growth of *E. coli* BL21. (A-D) Growth curves monitored in LB medium supplemented with 0, 0.1, 0.5, and 1 mM IPTG. (E) Comparison of growth profiles for the pYDT strain across the different IPTG concentrations. (F) Comparison of growth profiles for the pRS0121050 strain across the different IPTG concentrations. All experiments were performed in triplicate, and error bars represent the standard deviation (SD).

![Figure 2. A high-level overview of the concepts associated with bioelectrochemical systems.](https://static.igem.wiki/teams/5665/results/r3-2-1.webp)
![Figure 2. A high-level overview of the concepts associated with bioelectrochemical systems.](https://static.igem.wiki/teams/5665/results/r3-2-2.webp)

Figure 3-2 Growth curves of *E. coli* BL21 harboring pYDT (*E. coli* BL21/pYDT) and pRS0121050 (*E. coli* BL21/ pRS0121050) in LB medium under induction with varying concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG) are presented as follows: (A) 0 mM IPTG, (B) 0.1 mM IPTG, (C) 0.5 mM IPTG, and (D) 1 mM IPTG. Additionally, growth curves for strains (E) *E. coli* BL21/pYDT and (F) *E. coli* BL21/ pRS0121050 are compared across the different IPTG concentrations. The data are expressed as the mean of three independent replicates (n=3), with error bars indicating standard deviation (SD).

Based on these findings, we conclude that the PRK expression efficiency of the recombinant vector pYDT\_RS0121050 is higher than that of pYDT\_RS05100.

References

* [1] Carbajosa, S., Malki, M., Caillard, R., Lopez, M. F., Palomares, F. J., Martín-Gago, J. A., Rodríguez, N., Amils, R., Fernández, V. M., & De Lacey, A. L. (2010). Electrochemical growth of *Acidithiobacillus ferrooxidans* on a graphite electrode for obtaining a biocathode for direct electrocatalytic reduction of oxygen. *Biosensors & Bioelectronics, 26*(2), 877–880.
* [2] Kuhn, J., Müller, H., Salzig, D., & Czermak, P. (2015). A rapid method for an offline glycerol determination during microbial fermentation. *Electronic Journal of Biotechnology, 18(3), 252-255.*

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