Wet Lab Experiments

The experimental framework of Project POSEIDON formed the foundation of our biological validation process. This phase aimed to construct, transform, isolate, and verify the engineered genetic constructs responsible for metal-binding peptide synthesis, enabling the final assembly of the biosorption system. Each step was performed using aseptic techniques and standardized molecular biology protocols. The complete experiment spanned from August 30 to October 4, 2025.

Overview of Experiments

The wet lab experiments were structured into distinct phases covering reagent preparation, buffer optimization, competent cell preparation, transformation, plasmid extraction, restriction digestion, and verification. The following table provides a chronological overview of the performed experiments.

Plasmid Handling and Validation

The plasmids used in the study were procured from Dr. Vinay Bulusu’s lab. They included pET-M10, pET-28b, and pTWIST-Amp vectors. Each plasmid was verified through labeling inspection and resuspension in nuclease-free water. The cultures were revived on LB agar plates and monitored for contamination or unexpected growth patterns.

Preparation of Inoue Transformation Buffer (ITB)

The Inoue method provides highly efficient chemical competence by enhancing cell membrane permeability using divalent ions. The ITB was freshly prepared and filtered before use to maintain sterility. The reagents used and their concentrations are listed below.

Preparation of Competent Cells

Competent cells were prepared following the Inoue method. Pre-cultures were grown overnight and subcultured into fresh LB medium until OD₆₀₀ ≈ 0.4–0.5. Cells were harvested at 4°C, gently washed with ice-cold ITB, and resuspended with DMSO for cryoprotection. A total of 15 × 50 µL aliquots were snap-frozen in liquid nitrogen and stored at −80°C.

Transformation of Plasmids

The transformation was carried out using 50 µL aliquots of chemically competent E. coli cells. Each plasmid (pET-M10, pET-28b, pTWIST-Amp) was introduced using the standard heat-shock protocol. Post-recovery, cultures were plated on LB agar supplemented with the appropriate antibiotics (Kanamycin 50 µg/mL, Ampicillin 100 µg/mL). Colonies were visible after 16–18 hours of incubation at 37°C.

Plasmid Isolation

Miniprep of plasmid DNA was conducted using the alkaline lysis method. Each sample was eluted with pre-warmed nuclease-free water to ensure maximum yield. DNA concentration was measured using NanoDrop, recording an average of 201 ng/µL with a purity ratio (A260/280) close to 2.0. The following table summarizes the isolated plasmids.

Restriction Digestion and Gel Electrophoresis

Plasmids and inserts were subjected to double digestion using BamHI and HindIII to confirm fragment release. The reaction mixtures were incubated at 37°C for 1 hour and then electrophoresed on 0.8% agarose gel stained with SYBR Safe. Bands corresponding to ~220 bp for metallothionein (MT) and longer PCS fragments were observed. While most plasmids showed clear digestion patterns, faint bands in PCS lanes indicated partial enzyme efficiency.

Transformation of Gene Constructs

Post-verification, four constructs—MT-native, MT-engineered, PCS, and AtPCS—were transformed into pTWIST-Amp vectors for stable expression. Ampicillin selection (100 µg/mL) was used, and all constructs exhibited robust colony formation. Subsequent streak plating confirmed monoclonality, and glycerol stocks were prepared for preservation.

Final Outcomes

All essential plasmids and recombinant constructs were successfully transformed, isolated, and verified. The quality control checks ensured that each plasmid was free of contamination, with confirmed antibiotic resistance markers. These constructs form the basis for expression testing and downstream biosorption analysis. A summary of key outcomes from each experimental stage is given below.