Contributions

The contributions of the Metlock team are made possible through the efforts of fellow iGEMers globally. The work of past teams has laid a strong foundation of biological parts, software tools, and collaborative knowledge that continues to inspire and enable innovation in synthetic biology. This year's contributions are possible with gratitude to those who came before, whose efforts have shaped the opportunities available today.

The 2025 Metlock team has made four primary contributions to the iGEM Registry and community:

1. Biological Part: ZnuABC high-affinity zinc transporter [Zur Knockout]
2. Biological Part: AHL-LuxR induced ZnuABC high-affinity zinc transporter
3. Library of Biological Parts: ZnuABC[Zur Knockout]-Anderson Promoter Collection
4. A Custom Software Tools: Plot2Curve, a simulation that predicts mRNA and protein concentration over time under AHL induction and with Anderson family of promoters

The Metlock team has designed, constructed, and submitted a new composite BioBrick part encoding the ZnuABC high-affinity zinc transporter system. This three-gene transporter is naturally found in Escherichia coli and is essential for zinc uptake supporting many Zn²⁺-dependent proteins. We assembled the operon as a single cassette under a constitutive promoter for baseline characterization; the cassette is compatible with swapping to alternative promoters or inducible gates.

• ZnuA: periplasmic zinc-binding protein
• ZnuB: transmembrane permease
• ZnuC: cytoplasmic ATPase that powers transport

In E. coli, ZnuABC expression is normally governed by Zur, a zinc-responsive repressor that shuts down import once intracellular Zn²⁺ is sufficient. Disabling Zur control allows us to drive ZnuABC with synthetic regulators instead of the cell’s native sensor. This derepressed design decouples uptake from homeostatic feedback, enabling sustained, high-level import rather than automatic shutoff as zinc accumulates—ideal for biomining from dilute streams (e.g., industrial effluents, contaminated water). Once freed from native regulation, the cassette is modular: it can be placed under any Anderson promoter or an inducible scheme (e.g., LuxR/pLuxR), creating a versatile platform for tuning and future optimization.

Zinc is a vital cofactor for many enzymes, and its transport is tightly regulated. By providing the ZnuABC transporter as a ready-to-use BioBrick, we empower future teams to:

• Improve zinc uptake in engineered strains
• Design more efficient biosensors or bioaccumulation systems
• Study zinc transport dynamics under various environmental conditions
• Compare constitutive vs inducible control using a promoter ladder and AHL mapping to balance burden and performance

• Part Name: BBa_25V6WTD7
• Status: Submitted and available on the iGEM Registry
• Characterization: Still pending

Genetic Part: AHL-LuxR induced ZnuABC high-affinity zinc transporter

This part is our tunable zinc-uptake module and the core innovation of Metlock. We place the Zur-knockout ZnuABC operon under the BBa_K1893004 LuxR/pLuxR induction system, so ZnuABC expression is driven by AHL (acyl-homoserine lactone). Instead of one fixed uptake level, teams can dial expression up or down by changing AHL concentration. That makes it possible to find a “sweet spot” where zinc import is high without triggering zinc-induced stress.

Operon components:

• ZnuA: periplasmic zinc-binding protein
• ZnuB: membrane permease
• ZnuC: cytoplasmic ATPase that powers import
• BBa_K1893004: AHL-responsive LuxR/pLux induction cassette that drives the operon
• LuxR: AHL-dependent transcriptional activator for pLux
• AHL: small-molecule inducer that activates LuxR
• BBa_B0034: standard strong RBS placed before each ORF
• BBa_B0015: strong double terminator to stop transcription

• Why ditch Zur?: In E. coli, Zur represses ZnuABC when intracellular Zn²⁺ is sufficient. Removing Zur decouples the transporter from native feedback so we can control uptake externally.
• Why LuxR/pLuxR?: The system responds dose-dependently to AHL, giving a smooth input–output curve (Hill-type). By titrating AHL, teams can map inducer → expression and pick operating points tailored to their media or wastewater stream.
• Operational flexibility: Grow cells to high density uninduced (low burden), then add AHL only when you enter zinc-rich feed. This timing reduces stress during growth and maximizes metal capture during the production phase.

• Practical tuning instead of guesswork: AHL lets you adjust ZnuABC in small steps to match variable zinc availability across sites or days.
• Safety margin against toxicity: You can back off induction if cells show stress, or push higher when conditions allow.
• Comparable, reusable, modular: Any team can reuse the same inducible control, characterize their own AHL–response curve, and share conditions that work.
• Drop-in compatibility: The cassette slots into standard chassis and workflows, and can be paired with promoter ladders or additional logic if needed.

• Part Name: BBa_25J2UUD0
• Status: Submitted and available on the iGEM Registry
• Characterization: Still pending

Library of Genetic Parts: ZnuABC[Zur Knockout]-Anderson Promoter Collection

This library pairs the Zur-knockout ZnuABC zinc-uptake operon with the standardized Anderson promoter panel to create a constitutive “expression ladder.” It will come with a teachable, extensible measurement workflow: plate maps, SOPs (transform, grow, read), OD/fluorescence normalization steps, and analysis notebooks. This will allow teams to rerun the same pipeline on their strains and directly compare against our ladder once fully developed.

• Teachable, extensible measurement workflow: A ready-to-run template (plate maps, SOPs, normalization, notebooks) lets teams repeat the assay consistently and compare results across the ladder or across labs.
• Standardized benchmark for burden vs. benefit: Driving the Zur-KO ZnuABC cassette from weak → strong promoters yields fixed, reproducible transcription rates. Any team can quantify growth cost vs. uptake benefit on a shared scale, producing portable, comparable data.
• Inducer-free option for field deployments: Many deployments can’t rely on added inducers. Showing when constitutive ZnuABC is safe and effective provides a drop-in, no-inducer architecture for environmental or point-of-use devices.
• Direct bridge to inducible systems: Running the same cassette under AHL/LuxR and under Anderson promoters creates a clean constitutive vs inducible map (e.g., “expression for J23106 ≈ AHL at X nM”). Teams can choose between simplicity (constitutive) and control (inducible) and validate model predictions across both modes.

• With an sfGFP calibrant under each promoter, plate-reader units convert to relative promoter units (RPU) and then to absolute transcription rate (k_tx; RNAs·s⁻¹·cell⁻¹) for the chassis and media used.
• These calibrated rates slot directly into our promoter→mRNA→protein modeling tools, enabling dynamic-range predictions and recommended measurement windows for zinc assays.
• We will simulate in DBTL cycle 2, then verify in wet-lab cycles to produce reliable biological parameters for the models.

• Low (e.g., J23118–J23110): safe baseline capture, minimal burden.
• Mid (J23106–J23104): improved uptake with acceptable growth cost.
• High (J23100): maximum uptake; watch for membrane/assembly stress or cytotoxicity.

• Characterize across at least two plasmid backbones (low-copy pSC101, mid-copy p15A) and note chassis/media effects.
• Package data by promoter × backbone so results are reusable across common iGEM build contexts.

• Each “J231xx-ZnuABC [Zur Knockout]” part will include upon completed characterization: sequence, assembly notes, growth/uptake plots, RPU→k_tx calibration, and recommended use cases (e.g., “use J23110 for low-burden sensing”).
• We are publishing this library early as we troubleshoot our wet lab workflows so other teams can reuse the framework, extend it to related transporters, or collaborate on shared datasets.

Upon completion, we intent to release a set of final deliverables that will include a matched constitutive vs. AHL dataset (same cassette, same chassis),providing a rare, high-quality reference set for testing circuit models, codon-usage effects, and transporter stoichiometry hypotheses.

Collectively, these deliverables turn the Anderson–ZnuABC panel into more than another set of parts. It becomes a calibrated, field-relevant design space that teams can navigate to balance uptake performance against cellular health with clear guidance on when to choose constitutive vs. inducible control.

• Part Names:
  • J23100 – Synthetic ZnuABC [Zur Knockout] — BBa_25XGUUO0
  • J23101 – Synthetic ZnuABC [Zur Knockout] — BBa_25TMLF47
  • J23102 – Synthetic ZnuABC [Zur Knockout] — BBa_25325554
  • J23103 – Synthetic ZnuABC [Zur Knockout] — BBa_25GYPG97
  • J23104 – Synthetic ZnuABC [Zur Knockout] — BBa_252BBFCI
  • J23105 – Synthetic ZnuABC [Zur Knockout] — BBa_25Y94R5Y
  • J23106 – Synthetic ZnuABC [Zur Knockout] — BBa_25H1HJ61
  • J23107 – Synthetic ZnuABC [Zur Knockout] — BBa_25I8U9K2
  • J23108 – Synthetic ZnuABC [Zur Knockout] — BBa_25H1HJ61
  • J23109 – Synthetic ZnuABC [Zur Knockout] — BBa_25K3K03L
  • J23110 – Synthetic ZnuABC [Zur Knockout] — BBa_25WE9PXB
  • J23111 – Synthetic ZnuABC [Zur Knockout] — BBa_252U1CYF
  • J23113 – Synthetic ZnuABC [Zur Knockout] — BBa_25W2L367
  • J23114 – Synthetic ZnuABC [Zur Knockout] — BBa_25AIF769
  • J23115 – Synthetic ZnuABC [Zur Knockout] — BBa_25IFFIAD
  • J23116 – Synthetic ZnuABC [Zur Knockout] — BBa_25F5H5CX
  • J23117 – Synthetic ZnuABC [Zur Knockout] — BBa_25QJ5B1Z
  • J23118 – Synthetic ZnuABC [Zur Knockout] — BBa_25E3BDTN
• Status: Submitted and available on the iGEM Registry
• Characterization: Still pending