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.

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

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.

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.

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.

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.

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.

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.

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.