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.

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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.

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.

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.

Theoretically, the current-time curve was expected to exhibita 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.

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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 inA. 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.

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.

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

Test

The current-time curves were recorded, and thecurrent 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.

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

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.

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.

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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 thec-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.

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.

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.

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.

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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.

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.

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.

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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).

Test

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.

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.

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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.

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.

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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

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].

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.

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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.

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 theUniProt 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.

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.

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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.

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

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.

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.

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.