{
  "title": "Reteplase: change glutathione balance while holding DsbC fixed",
  "preparedOn": "2026-10-06",
  "status": "Submitted as a separate fourth design on October 6, 2026; no experimental results confirmed.",
  "reference": {
    "sampleId": "Reteplase_R2PC_041",
    "name": "Reteplase PEP (2026)",
    "url": "https://coopetition.htgaa.org/cfps",
    "composition": {
      "lysate": 5000,
      "template_reteplase": 2000,
      "base_buffer": 2000,
      "magnesium_glutamate": 225,
      "ammonium_glutamate": 200,
      "amino_acid_mix_17": 400,
      "hepes_koh": 550,
      "pep_mono": 6000,
      "folinic_acid": 50,
      "coa": 100,
      "nad": 75,
      "putrescine": 50,
      "spermidine": 125,
      "oxidized_glutathione": 475,
      "reduced_glutathione": 200,
      "potassium_oxalate": 150,
      "dsbc_ecoli": 1000,
      "atp": 250,
      "ctp": 175,
      "gtp": 175,
      "utp": 175,
      "nuclease_free_water": 625
    }
  },
  "existingSubmission": {
    "id": "q7u080ca30zs278",
    "url": "https://coopetition.htgaa.org/cfps?design=q7u080ca30zs278",
    "distinction": "The existing submission reduces DsbC to 3.75 uM while keeping the reference glutathione pair. This alternative restores the reference 5 uM DsbC and changes the glutathione pair. It has not replaced the existing submission."
  },
  "question": "Does changing the added glutathione balance change functional Reteplase output when the course PEP mixture and DsbC are otherwise held fixed?",
  "evidenceGap": "All 312 experimental wells in the inspected Reteplase control plate used the same glutathione and DsbC additions. That plate cannot resolve this particular contrast.",
  "whyThisQuestion": "Yin and Swartz (2004) used extract treatment and an oxidized/reduced glutathione mixture when producing an active nine-disulfide plasminogen activator in an E. coli cell-free system. This supports investigating folding conditions. Their combined improvements do not establish an optimum for this course lysate or predict this candidate's result.",
  "hypothesis": "Redistributing the added glutathione pool toward GSH could alter the balance of disulfide formation and rearrangement. It could increase, decrease, or leave functional output unchanged. The experiment tests a composition contrast, not a known improvement.",
  "changes": [
    {
      "reagent": "nuclease_free_water",
      "referenceNl": 625,
      "alternativeNl": 325
    },
    {
      "reagent": "oxidized_glutathione",
      "referenceNl": 475,
      "alternativeNl": 350
    },
    {
      "reagent": "reduced_glutathione",
      "referenceNl": 200,
      "alternativeNl": 625
    }
  ],
  "concentrationComparison": {
    "reference": {
      "addedGSSGmM": 4.0375,
      "addedGSHmM": 1,
      "DsbCuM": 5,
      "addedGlutathioneEquivalentsmM": 9.075,
      "addedGSHtoGSSGMolarRatio": 0.24767801857585142
    },
    "alternative": {
      "addedGSSGmM": 2.975,
      "addedGSHmM": 3.125,
      "DsbCuM": 5,
      "addedGlutathioneEquivalentsmM": 9.075,
      "addedGSHtoGSSGMolarRatio": 1.050420168067227
    }
  },
  "arithmetic": [
    "Reference GSSG: 170 mM × 475/20,000 = 4.0375 mM; reference GSH: 100 mM × 200/20,000 = 1 mM.",
    "Alternative GSSG: 170 mM × 350/20,000 = 2.975 mM; alternative GSH: 100 mM × 625/20,000 = 3.125 mM.",
    "Glutathione equivalents: reference 1 + 2×4.0375 = 9.075 mM; alternative 3.125 + 2×2.975 = 9.075 mM.",
    "Net stock-volume increase is 425 - 125 = 300 nL; water changes from 625 to 325 nL."
  ],
  "comparisonPlan": [
    "Run the alternative and unchanged Reteplase_R2PC_041 reference together with the same lysate lot, target template, incubation conditions, and assay timing. Replicate and distribute conditions across plate positions.",
    "Use the course activity assay with suitable matrix blanks and calibration. Check that changing the reaction matrix does not itself alter the activity readout, for example with matched spike recovery or a dilution check.",
    "Measure soluble target alongside activity if available. Record pH; repeat a promising contrast in another lysate lot before making a robustness claim."
  ],
  "interpretation": {
    "higherActivity": "Would support using this glutathione composition under the tested conditions. Activity alone cannot separate more expressed protein, greater soluble recovery, altered specific activity, or assay-matrix effects.",
    "unchangedActivity": "Would show no resolved functional benefit for this tested contrast and assay precision. It would not establish that all redox changes are ineffective.",
    "lowerActivity": "Would argue against this particular shift under the tested conditions. It would not prove a single failure mechanism or establish the reference as globally optimal."
  },
  "limits": [
    "The constant quantity is added [GSH]+2[GSSG], expressed as glutathione-monomer equivalents. The sum [GSH]+[GSSG], free thiol concentration, and total solute-particle concentration are not held constant.",
    "The stock additions do not determine the actual reaction redox potential. pH, lysate components, other redox couples, and changes over time matter; no numerical potential is asserted.",
    "The paired GSSG/GSH adjustment is one planned contrast but changes two stock volumes. A single comparison cannot map the full redox response or prove a disulfide-folding mechanism.",
    "No measured yield, protease activity, or cost-per-activity is available for this alternative. The selected doses are an experimental contrast, not a validated optimum.",
    "DsbC is kept at the course-stated 5 uM. No independent monomer-versus-dimer concentration convention is assumed."
  ],
  "priorExperimentAudit": {
    "checkedOn": "2026-10-06",
    "source": "https://coopetition.htgaa.org/cfps/__data.json",
    "localEvidence": "tmp/reteplase-data-decoded.json",
    "run": "reteplase_control_20260730-DSB",
    "experimentalWellsChecked": 312,
    "allPublishedExperimentalWells": {
      "dsbc_ecoli": 1000,
      "oxidized_glutathione": 475,
      "reduced_glutathione": 200
    },
    "exactAlternativeMatchFound": false,
    "scope": "The published control plate captured on October 6, 2026. This does not establish uniqueness across all historical experiments or other students' submissions."
  },
  "sources": [
    {
      "title": "Course CFPS reference and reagent inventory",
      "url": "https://coopetition.htgaa.org/cfps",
      "checkedOn": "2026-10-06"
    },
    {
      "title": "Published course control-plate data",
      "url": "https://coopetition.htgaa.org/cfps/__data.json",
      "checkedOn": "2026-10-06",
      "localEvidence": "tmp/reteplase-data-decoded.json"
    },
    {
      "title": "Yin & Swartz (2004), Enhancing multiple disulfide bonded protein folding in a cell-free system",
      "url": "https://pubmed.ncbi.nlm.nih.gov/15052638/",
      "doi": "10.1002/bit.10827",
      "evidenceRead": "Primary-paper abstract, checked October 6, 2026."
    }
  ],
  "aiDisclosure": {
    "client": "Codex (OpenAI); exact model version not recorded",
    "sourceExample": "site/examples/wet-lab-expert/project.json",
    "sourceRevision": 4,
    "currentTaskContext": "The user approved preparing experiment briefs and a Reteplase redox-balance alternative as part of a learning-focused homework plan.",
    "preparation": "Codex extracted the existing simulated wet-lab-expert candidate, checked its course-model export and volume math, audited the cached published control plate, and wrote this brief. Codex submitted the fourth design with user authorization on October 6, 2026. No new laboratory experiment or measured yield is claimed."
  },
  "submission": {
    "id": "895s12z492g2nxz",
    "sampleId": "ilknur_icke_2026-10-06_reteplase_pep_glutathione_balance_v1",
    "url": "https://coopetition.htgaa.org/cfps?design=895s12z492g2nxz",
    "submittedOn": "2026-10-06",
    "confirmation": "The course site displayed: Submitted composition — saved as 895s12z492g2nxz."
  }
}