Engineering Success
Introduction
Mining and metal recovery are essential to modern industries, but conventional methods are energy-intensive, environmentally destructive, and generate toxic byproducts [1–3]. Metlock aims to develop a safe, efficient biomining system using engineered E. coli to selectively gather zinc from ores, industrial waste, and polluted environments/water sources. By synthetically regulating high-affinity zinc transport systems, Metlock demonstrates an iterative engineering approach to create a modular platform for sustainable metal recovery [4–6].
Metlock took on a challenging set of design goals: develop a library of synthetic genetic parts useful in biomining and remain modular for future contributions. Our final objective is for Metlock to expand to encompass several designer strains targeted for a variety of metals, accomadating both our current amount of talent and resources, this year we focused on the following design goal: design a fine-tunable system to maximize zinc influx while avoiding cytotoxicity. This will allow us to pilot workflows, develop our skills with the necessary molecular methods, and expand our dry lab skillset for our more advanced project goals. Following strain identification, this system could then be tuned for profitable yield in-lab and eventually deployed in simulated industrial settings.
Design
To implement our goal of a tunable zinc uptake system, E. coli’s endogenous ZnuABC operon was redesigned in-silico to no longer be repressed by Zur and placed under the induction of the BBa_K1960004 cassette – a part originally designed by Shijia Qi 2016 [7,4,6]. This allows for the tuning of expression of ZnuABC across an AHL gradient to tune expression as well as across copy number in clonal vectors [8–9,10]. The individual components of ZnuABC serve the following functions: ZnuA is the periplasmic solute-binding protein that captures Zn²⁺, ZnuB is the inner-membrane permease that translocates Zn²⁺, and ZnuC is the cytosolic ATPase that powers transport by hydrolyzing ATP [5].
Downstream of pLuxR, ZnuABC is expressed only in the presence of AHL, tying expression of this active metal transporting complex to the formation of AHL/LuxR complexes [8–9,10]. In this configuration, the titration of AHL into media containing bacteria transformed with this iteration of Metlock would theoretically lead to tunable expression of znuABC.
To validate the tunable zinc import system, we plan to utilize a colorimetric uptake assay using the Zn-specific dye Zincon (Z002), which forms a blue Zn–complex measurable at λ_max = 620 nm (ε ≈ 23,000 M⁻¹cm⁻¹) under alkaline conditions (pH ~9) [11].
RECOMMENDED ASSAY: Zincon-based Zn²⁺ uptake (supernatant-depletion readout)
Goal: Quantify Zn²⁺ import as a function of AHL induction of your Zur-independent iZnuABC cassette (LuxR/pLuxR control) using a colorimetric readout of residual extracellular Zn²⁺ with Zincon at λₘₐₓ ≈ 620 nm in pH 9 buffer [11,12,13,14].
Rationale: ZnuA/B/C is the high-affinity ABC Zn²⁺ importer (ZnuA: periplasmic binder; ZnuB: permease; ZnuC: ATPase), and Δznu mutants show impaired Zn acquisition phenotypes under Zn limitation which is ideal for controls [15,16,17,5].
Strains:
- iZnuABC (LuxR/pLuxR-driven, Zur-independent znuABC).
- ΔznuB knockout (negative control for transport, this would be a strain of synthetic ZnuABC which does not contain ZnuB).
Empty-vector control.
(pLuxR/LuxR cassette ref: BBa_K1960004 [7,8].)
Reagents & prep:
- ZnSO₄ stock (e.g., 10 mM).
- Metal-free water/buffers/media prepared by batch treatment with Chelex-100 and thorough rinsing [18–20].
- AHL gradient (e.g., 0, 1, 3, 10, 30, 100, 300 nM) to span induction range [7,8].
- Zincon reagents and pH 9 buffer for the spectrophotometric method (Standard Methods 3500-Zn B; λ = 620 nm; includes optional cyanide/cyclohexanone masking for interferences) [12,11,13].
For a cyanide-free workflow in clean matrices, use a straightforward microplate adaptation at pH 9 with calibration and spike-recovery checks [11,14].
Do not add EDTA to measurement tubes; it chelates Zn²⁺ and will depress the Zincon signal [21].
Culture & induction:
Grow strains in Zn-depleted M9 (Chelex-treated) overnight; back-dilute 1:100 into fresh Zn-depleted M9 ± AHL; grow to OD₆₀₀ ≈ 0.3–0.4, then induce/express iZnuABC for ~90–120 min; wash and resuspend in Zn-free uptake buffer to OD₆₀₀ = 0.5 [7,8,18–20].
Uptake reaction (time-course):
Mix cells with 25–50 µM ZnSO₄ in uptake buffer (e.g., 1.0 mL tubes or 200 µL wells at 30–37 °C).
Collect cell-free supernatant at 0, 2, 5, 10, 20, 30, 45, 60 min (quick pellet or filter). Keep on ice. (No EDTA in these aliquots.) [12,11,13,21]
Zn²⁺ measurement (Zincon @ 620 nm):
- Path A: Standard Methods 3500-Zn B (robust): buffered pH 9; sodium ascorbate to suppress Mn interference; CN⁻ to complex metals; cyclohexanone to release Zn selectively; read 620 nm. Use only in labs approved for cyanide handling [12,13]
- Path B: Cyanide-free microplate (clean matrices): pH 9 buffer + Zincon; read 620 nm; verify matrix with spike-recovery ≥ 90% and note potential Cu interference [11,14].
Controls:
ΔznuB knockout (low uptake), empty vector, heat-killed cells (adsorption), no-cell blank, Zn standards (5–100 µM; R² > 0.99), and spike-recovery samples; optional post-run EDTA “surface-strip” on pellets (not for Zincon readout) [12,11,13,16,17,5,21].
Calculations & output:
Convert A620 to [Zn] via the standard curve; compute Uptake(t) = ([Zn]₀ − [Zn]ₜ)×V; normalize by OD₆₀₀ (or mg protein). Report initial rate (0–10 min slope; pmol Zn·OD₆₀₀⁻¹·min⁻¹) and plot uptake vs AHL to obtain induction curves (Hill fit optional) [11,12,13,14].
Hypothesized results:
Induced iZnuABC shows AHL-dependent increases in uptake; ΔznuB remains near baseline; empty-vector minimal. This pattern reflects high-affinity ZnuABC transport under Zn limitation [5,15,16,17].
Build
Clonal Vector Ordering: We ordered our parts using the clonal vectors offered by the Twist Bioscience iGEM Sponsorship, cloning our iZnuABC (LuxR/pLuxR-driven, Zur-independent znuABC) into the pUPD3 vector [15, 16].
Transformations: We transformed the plasmid by heat-shock into E. coli BL21, DH5α, and TOP10 strains as test chassis; our pUC19 positive-control transformation succeeded, but we observed no expression from the ordered clonal vector from its antibiotic resistance cassette - resulting in no viable cells to continue our experiments with [17–23].
Test
Objective: Demonstrate that LuxR/pLuxR-induced, Zur-independent iZnuABC increases Zn²⁺ import, quantified by Zincon supernatant-depletion across an AHL gradient [11–14,5,15–17].
Observed block: pUC19 transformed successfully, but no colonies from the pUPD3::synthetic ZnuABC clonal vector on chloramphenicol plates (correct selection for pUPD3 is Chloramphenicol) [22].
Most likely causes (with fixes):
- Transformation workflow sensitivity (outgrowth): Chloramphenicol is bacteriostatic; giving ~60 min outgrowth in SOC at 37 °C with shaking materially improves survival/expression of the cat (chloramphenicol acetyltransferase) marker before plating [30,34].
- Antibiotic working range: Plate a Chloramphenicol gradient (12.5 / 25 / 34 / 50 µg/mL) in parallel; 25 µg/mL is the standard recommendation for cat-based backbones like pUPD3 [22,31].
- Plasmid size/quality: Large assemblies or salt-contaminated DNA reduce chemical-transformation efficiency; try electroporation (1–2 mm cuvette, ~1.8 kV, ~5 ms pulse) which boosts efficiency for bigger plasmids [35,34].
- Vector verification: Run restriction digest/agarose on the clonal DNA and PCR across backbone and insert junctions after a small-scale no-antibiotic recovery (patch survivors to Chloramphenicol) to confirm integrity before concluding a build error [22,34].
- Strain idiosyncrasies: Keep DH5α/TOP10 as cloning strains; BL21 is fine but not necessary at this step. If chemical competence is borderline, prepare fresh Hanahan/Inoue-style competent cells or use commercial high-efficiency cells [36–38].
QC gates to unlock the assay
- Replica plating check: Transform pUPD3::iZnuABC and plate across the Chloramphenicol gradient + a no-antibiotic plate; incubate 18–24 h. Positive hits should appear ≥12.5–25 µg/mL if the cat cassette is intact [22,31,30].
- Colony PCR + Sanger: Verify (1) backbone and insert and (2) znuA, znuB, and znuC junctions in 2–4 colonies. Archive glycerol stocks [22].
- (Optional) Induction sanity-check: Parallel strain carrying pLuxR-reporter to locate a clean AHL window before metal assays [8–10].
Functional readout (after QC passes): Perform the Zincon uptake assay as specified (Zn-depleted medium, AHL 0–300 nM, 25–50 µM ZnSO₄, supernatant @ 620 nm in pH 9 buffer; exclude EDTA from read tubes; include ΔznuB and empty-vector controls) and report initial uptake rate (0–10 min slope; pmol Zn·OD₆₀₀⁻¹·min⁻¹) and AHL dose–response [11–14,18–21].
Expected outcome: With verified constructs on Chloramphenicol and a confirmed induction window, iZnuABC shows AHL-dependent increases in uptake; ΔznuB remains near baseline; empty-vector minimal [5,15–17].