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Yeast UOX Expression Cassette — Matched Build and Measurement Plan

Scope

Define sequence-controlled, matched Saccharomyces UOX constructs that can distinguish effects of payload identity, synonymous coding sequence, promoter, terminator, copy strategy, substrate access, and topology.

This page does not recommend a sequence variant, promoter, terminator, vector, integration strategy, topology, expression target, formulation, or dose.

Sequence Provenance

For the Aspergillus flavus UOX reference:

  • Protein record: UniProt Q00511.
  • Coding-sequence record: GenBank X61766.1, 906 bp.
  • Q00511 contains a 302-residue precursor; the annotated mature chain after initial-methionine removal is 301 aa. Record explicitly whether construct and assay numbering include the initiator methionine.
  • P78609 is the Candida utilis/Cyberlindnera jadinii UOX record, not the A. flavus record. Treat it as a separate payload identity routed through variant selection.

Every construct record must include the exact nucleotide sequence, translated sequence, accession and version, leader or anchor junctions, stop codon, and numbering convention. A name such as “uaZ,” “wild type,” or “codon optimized” is not sufficient provenance.

Design Matrix

Use matched pairs or a preregistered factorial. Do not change promoter, terminator, copy strategy, host background, and topology simultaneously and then attribute the result to one element.

Axis Candidate comparison What remains fixed
Payload Exact UOX sequences selected through variant selection Host, topology, promoter, terminator, copy strategy, process, assay
Synonymous coding sequence Source CDS and preregistered synonymous variants Amino-acid sequence and all noncoding elements
Promoter Defined constitutive and inducible candidates UOX sequence, topology, terminator, copy strategy, locus or vector
Terminator Defined terminator candidates UOX sequence, topology, promoter, copy strategy, locus or vector
Copy strategy Defined plasmid or chromosomal-integration candidates Cassette sequence, host background, process, assay
Topology No leader; defined secretion leader; defined surface anchor UOX sequence, promoter, terminator, copy strategy, host, process
Substrate access No added transporter; defined transporter; transport-inactive control UOX cassette and assay conditions

Candidate part names may include TDH3, PGK1, TEF1, or GAL1 promoters; CYC1, ADH1, or CPS1 terminators; and plasmid or defined chromosomal-integration strategies. These are build options, not ranked choices.

Matched Topology Schemas

Intracellular comparator
promoter ─ UOX ─ terminator

Secreted comparator
same promoter ─ defined secretion leader ─ same UOX ─ same terminator

Surface-displayed comparator
same promoter ─ defined leader/anchor ─ same UOX ─ same terminator

A recovered cell-free fraction is a process comparator rather than a distinct expression cassette. Measure it from the matched producing construct and record every recovery step.

Sequence and Build Acceptance

  • Sequence-verify the complete cassette and every junction.
  • Confirm the translated UOX sequence and numbering convention against the pinned source record.
  • Record all synonymous changes and the constraint that motivated each one.
  • Record predicted codon, GC, motif, and RNA-structure descriptors as design metadata only.
  • Verify the integration locus or plasmid identity, orientation, and copy number.
  • Record host background and passage history.
  • Use an inactive-UOX counterpart and a chassis-only control in the same cassette context.

Codon scores and expression forecasts are not acceptance criteria. A synonymous variant advances only through measured soluble and active UOX under matched conditions.

Measurement Panel

Layer Required readouts
Construct Complete sequence, junctions, locus or vector, orientation, copy number
Transcription Transcript abundance when needed to localize failure
Protein Total UOX, soluble UOX, oligomeric state, degradation products
Localization Intracellular, surface-associated, and extracellular UOX measured separately
Activity Active IU in each fraction with assay substrate, pH, oxygen, time, and uncertainty reported
Host Growth, viability, genetic stability, and batch variance
Process Active UOX before and after recovery, storage, formulation, and simulated transit steps actually proposed
Reaction site Urate, reaction product, dissolved oxygen, hydrogen peroxide, UOX persistence, and viability over time

Report total protein and active UOX separately. Do not convert promoter identity, codon score, transcript abundance, or total protein into a delivered dose.

Decision Sequence

  1. Identity: Reject any construct whose sequence, junctions, host background, or copy state is unresolved.
  2. Active expression: Advance only constructs with reproducible active UOX, interpretable localization, acceptable viability, and batch variance reported.
  3. Topology: Expression measurements nominate configurations for validation §1.33; they do not select a topology. A yeast arm must be explicit in §1.33 or in a preregistered matched extension using the same control and decision framework.
  4. Safety: A §1.33 survivor must clear validation §1.36.
  5. Escalation: No animal design follows until both gates pass.

If a construct fails, change the smallest axis supported by the failure localization, rebuild the matched control, and repeat the relevant measurement. Do not rescue a weak result with an unmeasured yield, dose, survival, or substrate-access assumption.

Evidence Boundary

  • Leplatois et al. establish In Vitro intracellular active A. flavus UOX expression in S. cerevisiae. They do not select the present cassette or oral topology.
  • Wang et al. establish In Vitro engineered yeast uric-acid catabolism and show that payload, transporter, promoter, and integration context can be tested experimentally. Their result does not select this project's construct.
  • Mechanistic Extrapolation: COMP-019's unconditional flat-dose classification is not robust to COMP-044's tested substrate-occupancy and finite-window diagnostics. COMP-044 supplies no expression or production-sufficiency target, replacement ΔSUA, dose, genotype order, physiological regime, efficacy model, topology/chassis selection, or safety conclusion.

Primary Sources and Decision Records