Week 1 HW: Principles and Practices

Zambia Mineral-Waste Bioremediation Predictor

From Metagenome to Marketable Bioremediation Product

HTGAA 2026 Final Project · Elsa Muleya · SynBio USFQ Node

Project Rationale

Zambia’s Copperbelt Province faces severe heavy metal contamination from decades of copper mining at Konkola, Nchanga, Mufulira, and Chingola. Cu²⁺, Zn²⁺, Co²⁺, and Pb²⁺ leach from mine tailings into groundwater and agricultural soils at concentrations far exceeding WHO limits, with no affordable or accessible remediation solution for affected communities.

This project designs, validates, and packages a living biological solution: engineered Bacillus subtilis carrying a novel metallothionein (MT) gene discovered from Zambian mine-associated bacterial genomes, encapsulated in a field-deployable dual-layer hydrogel biocontainment system — ZAMGEL — that can be commercially produced and applied without specialist equipment or laboratory infrastructure.

Three-Aim Project Structure

Aim 1: Bioinformatics Discovery & Genetic Design

Goal: Identify and structurally validate novel metallothioneins from Zambian mine-associated bacterial genomes, and design a complete synthetic expression cassette ready for wet lab transformation.

Sub-aim 1a: Metagenomic Mining of Zambian Copperbelt Sequences

Mine publicly available sequencing datasets from NCBI SRA, MG-RAST, and IMG/M targeting the Konkola, Nchanga, and Mufulira mine regions. The full computational pipeline:

FASTQ → fastp (QC trim) → MEGAHIT (assembly) → Prodigal (ORF prediction) → BLASTp + Prokka (annotation)

Filter candidates by the presence of the Cys-X-Cys motif — the canonical Cu/Zn coordination fingerprint in prokaryotic metallothioneins — and cross-reference against known prokaryotic MT families (SmtA-like, BmtA-like, CzcA operons, CopA ATPases). Build a maximum-likelihood phylogenetic tree using IQ-TREE 2 to confirm novelty.

Sub-aim 1b: Structural Validation & Synthetic Expression Cassette Design

For the top 5 MT candidates from Sub-aim 1a, simultaneously validate 3D structural integrity and design the full synthetic genetic system.

Structural Validation

• Submit top candidate sequences to AlphaFold3 to generate .pdb files and visualise cysteine-rich metal-binding pockets
• Pass threshold: pLDDT > 85 across the metal-binding domain; ipTM > 0.80 for confident fold prediction
• Quantify binding pocket geometry in PyMOL / ChimeraX: pocket volume (ų), solvent accessibility, Cys coordination angle, and closest Cys–Cys distance (target < 6 Å for effective Cu²⁺ coordination)
• Calculate predicted dissociation constant: Kd = e^(ΔG/RT) at T = 310 K (37°C); expected range 10⁻¹³ to 10⁻¹⁵ M for high-performance prokaryotic MTs
• Compare all candidates against reference proteins (SmtA from Synechococcus PCC 7942; BmtA from Pseudomonas) on Kd, Cys count, and pLDDT

Expression Cassette Design (Benchling)

• Codon-optimise the best-scoring MT sequence for B. subtilis 168 using Benchling’s built-in optimiser
• Design a metal-responsive synthetic circuit in Cello 2.0: Cu²⁺ sensor (PcorA or PmtA promoter) → NOT gate logic → MT expressed only when Cu²⁺ exceeds threshold
• Include eGFP fluorescent reporter downstream of MT as a real-time visual proxy for circuit activation

5'─[PcopA/PmtA]─[RBS B0034]─[MT_Bsubtilis_optimised]─[eGFP]─[T_B0015]─3'
    Cu²⁺ sensor   strong RBS    codon-optimised         reporter  terminator

• Verify BioBrick RFC10 compatibility in Benchling
• Submit all sequences through Twist Bioscience biosecurity screening (“Green” classification required before synthesis order)

Aim 2: Wet Lab Validation Under Zambian Environmental Conditions

Goal: Transform the computationally designed system into a living, functional biosensor-remediator and rigorously stress-test it against the real environmental conditions of the Zambian Copperbelt.

Sub-aim 2a: Chassis Construction & Verification

Transform B. subtilis 168 with the assembled MT expression plasmid and confirm successful integration using three independent assays before proceeding to metal exposure experiments:

Sub-aim 2b: Metal Ion Concentration Response Assays

Expose the engineered B. subtilis to a full Cu²⁺ concentration gradient spanning real Copperbelt mine drainage (reported range: 0.5–500 mg/L). Measure metal removal using ICP-MS on growth media supernatant and calculate Bio-Sequestration Efficiency (%BSE):

%BSE = ([Metal]₀ − [Metal]f) ÷ [Metal]₀ × 100

Sub-aim 2c: pH Stress Testing

Zambian mine tailings range from pH 2.5–4.5 (active acid mine drainage) to pH 8–9 (alkaline neutralisation runoff). Test bacteria across this full range at fixed 50 mg/L Cu²⁺ to define the operational pH window and inform ZAMGEL outer shell buffer design.

Sub-aim 2d: Multi-Stressor Environmental Simulation

Real Copperbelt soil presents multiple co-occurring stresses. Bacteria must survive all of these simultaneously to be field-deployable. Each stressor is tested at fixed Cu²⁺ = 50 mg/L and pH 6.5 to isolate the effect; a final cocktail experiment combines all worst-case stressors simultaneously.

Aim 3: ZAMGEL Containment System & Commercial Product Design

Goal: Design a biomaterial containment system that physically and genetically contains the engineered bacteria inside a field-deployable carrier, preventing environmental escape while maintaining full metal-sequestration function — creating a product that can be commercially sold and applied without ecological risk.

Sub-aim 3a: ZAMGEL Dual-Layer Hydrogel Bioencapsulation

The ZAMGEL biocapsule is a three-layer biomaterial architecture. Each layer performs a distinct function, together creating a self-contained living bioreactor deployable directly onto mine tailings:

Sub-aim 3b: Containment Validation & Kill-Switch Integration

Containment Validation

Genetic Kill-Switch (MazF/MazE Toxin-Antitoxin)

A MazF/MazE kill-switch is integrated into the B. subtilis chromosome (not plasmid, to prevent loss). MazE antitoxin is expressed under a Ptet promoter requiring anhydrotetracycline (aTc) to remain active. When aTc is withdrawn (ZAMGEL retrieved or degraded at end of life), MazE degrades, MazF mRNA interferase cleaves all mRNA, and all bacteria die within 48 hours. A secondary CcdB/CcdA kill-switch on the plasmid backbone provides an orthogonal safety layer.

aTc present → MazE expressed → MazF neutralised → Bacteria LIVE
aTc absent  → MazE degraded  → MazF active      → Bacteria DEAD within 48h

Sub-aim 3c: Commercial Product Formats & Digital Predictor App

A Streamlit-based mobile web app (offline-capable PWA) allows community members and mine site managers to input local soil Cu²⁺ concentration, pH, temperature, and treatment area, and receive a data-driven treatment recipe — number of ZAMGEL beads, predicted %BSE, and estimated remediation timeline — based on dose-response curves generated in Aim 2. No laboratory equipment required.

Regulatory pathway: Zambia Environmental Management Agency (ZEMA) contained-use application under Biosafety Act No. 10 of 2007; Nagoya Protocol compliance for use of indigenous Zambian microbial genetic resources; community consent framework with Copperbelt mining communities. Primary commercial client: ZCCM-IH.

15-Week Project Timeline

Validation Criteria & Contingency Plans

Why This Project Matters

Existing Copperbelt remediation approaches — lime neutralisation, chemical precipitation, pump-and-treat — are capital-intensive, infrastructure-dependent, and inaccessible to subsistence communities adjacent to mine tailings. The ZAMGEL system offers:

• No electricity or specialist infrastructure required — scatter-and-forget deployment
• Zero environmental release — physically contained by 200 nm membrane; genetically contained by dual kill-switch
• Self-regulating — MT only expressed when Cu²⁺ exceeds threshold; GFP reporter confirms activity in real time
• Locally grounded — MT gene discovered from Zambian mine-associated bacterial genomes
• Commercially viable — manufacturable from locally sourced materials; approvable under existing Zambian biosafety law
• Community-facing — Streamlit app enables treatment planning without laboratory equipment or expertise