Week 3 HW: Cell-Free Reaction Design

Ilknur Icke · HTBAA Fall 2026 · October 6, 2026 · AI-assisted

Introduction

Watch the introduction from CFPS Design Studio I am working on building (8 minutes 27 seconds).

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I initially submitted one reaction composition for each target: sfGFP, PETase/TfCut2 and Reteplase. My first round uses small changes to tested course references. After comparing the proposals with the published experiments, I decided to emphasize what each experiment could teach us, as well as its possible cost benefit. I then submitted a fourth design to test a different glutathione balance in the Reteplase reaction. Its recipe and experimental question are below.

The answers below were prepared with AI assistance and checked against the cited sources. Preparing the text does not replace being able to explain the reasoning at recitation. Assignment brief · Course builder.

A1 · Why do salt concentrations differ, and which is more sensitive?

Potassium and magnesium concentrations differ because each formulation creates a different chemical environment. Potassium helps support the ionic conditions needed for protein–nucleic acid interactions. Magnesium supports the translation machinery and binds nucleotides and other metabolites, so changing the energy system, nucleotide concentrations or lysate can change the magnesium requirement. Added magnesium is not the same as free magnesium.

In the PANOx-SP salt titration, magnesium was more sensitive over the concentrations tested: increasing magnesium glutamate from 20 to 24 mM reduced relative CAT expression from 1.00 to 0.73, whereas increasing potassium glutamate from 175 to 200 mM reduced it from 1.00 to 0.94. These are separate titrations with different concentration scales. They support adjusting magnesium carefully, rather than assuming a universal optimum. The Table II titration values exclude extract carryover; the extract supplies approximately another 3.3 mM magnesium and 14.4 mM potassium. Jewett & Swartz (2004), Table II and its footnotes, journal p. 22.

For a new reaction, I would retest magnesium when changing the energy or nucleotide formulation. The CAT experiment explains why this matters; it does not establish the optimum for sfGFP, TfCut2 or Reteplase.

A2 · How does PEP + NTP differ from ribose + NMP?

PEP + NTP starts with nucleotide triphosphates (ATP, CTP, GTP and UTP), which supply RNA building blocks; ATP and GTP also support energy-consuming steps. PEP provides a phosphate-containing energy substrate to help replenish ATP. With ribose + NMP, the starting nucleotides have one phosphate rather than three. The lysate must turn them into usable triphosphates, supported by its metabolism and the full energy mixture.

Olsen et al. (2026), Results and Figure 2, found sustained ATP in their optimized formulation without added ATP. That system was more sensitive to reaction geometry and oxygen supply. Their work shows that a cheaper nucleotide strategy can work, but a one-for-one ingredient swap would not recreate it (https://www.nature.com/articles/s41467-026-69605-8#Fig2).

I would first establish a PEP/NTP baseline and then compare a separately specified ribose/NMP formulation using the documented course stocks and base-buffer contributions. The comparison should use the same protein, lysate batch, vessel and assay. The useful economic result is cost per functional protein output, so lower reagent prices alone would not decide the winner.

A3 · Compare two published compositions

I compared PANOx-SP from Jewett and Swartz with Olsen’s RFopt. They both provide an E. coli extract with the materials needed to make protein, but they make different choices about how to supply energy and RNA building blocks.

The numbers come from Jewett & Swartz (2004), Table I and Methods, PDF p. 2 and Olsen et al. (2026), Table 1, Methods and Supplementary Tables 1–2. This table focuses on the ingredients most relevant to the comparison; the cited papers contain the complete formulations. The salt entries above are the named formulation inputs. PANOx-SP also carries 3.3 mM magnesium acetate and 14.4 mM potassium acetate from the extract. A magnesium-glutamate value alone therefore does not describe all the magnesium in the reaction, and none of these values measures free magnesium.

PANOx-SP supplies RNA-ready triphosphates and uses PEP as an energy substrate. RFopt starts with monophosphates, which have fewer phosphate groups. The reaction’s metabolism must convert them into the triphosphates used for RNA production. Ribose, glucose and phosphate are parts of that whole system. I would not reproduce RFopt by swapping only PEP for ribose in an otherwise unchanged PANOx-SP reaction.

RFopt uses less magnesium glutamate and more potassium glutamate in the listed additions. That shows why an ingredient’s concentration should be considered with the rest of the mixture. Its higher HEPES concentration is another substantial change. PANOx-SP also includes NAD, coenzyme A, folinic acid and added tRNA in its Methods recipe. An ingredient absent from RFopt’s listed additions can still be present in the extract; absence from a recipe is not proof that the reaction never needs that molecule.

Olsen reports reagent costs of $143 per litre of reaction, or $60 per gram of sfGFP, for RFopt under the paper’s pricing and yield assumptions. Those prices exclude DNA and lysate. I cannot calculate a fair cost advantage over the 2004 PANOx-SP recipe without pricing both recipes on the same basis.

The experiments also differ: Jewett and Swartz used CAT and A19-derived extract at 37 °C, whereas Olsen’s standard experiments used sfGFP and a different extract at 30 °C for 20 hours. Their vessels and volumes also differ. I therefore conclude that RFopt offers a documented alternative way to supply nucleotides and energy, rather than claiming that this table proves which recipe will work best for the homework’s proteins.

These are the published formulations; the course’s PEP and ribose references use its own stocks, fixed inputs and experimental conditions. Olsen’s PANOx-SP comparison column also differs from the original 2004 recipe, so I do not use it to assign a modern price to the original formulation.

B1 · The initial three submitted designs

Each reaction totals 20,000 nL. It contains 5,000 nL lysate, 2,000 nL target DNA and 2,000 nL fixed base buffer. Reagent additions and water occupy the remaining 11,000 nL. Every transfer is a whole multiple of 25 nL.

The documented base buffer contributes 200 mM potassium glutamate, 2.6 mM magnesium glutamate, 30 mM HEPES and 1 mM of each of the 17 amino acids, plus tyrosine and cysteine. The concentrations below add the specified buffer contribution to the supplemental stock contribution. They do not measure free magnesium or all molecules carried in lysate. Recipes use PEP mono at 100 mM, not the separate 500 mM cyclohexylammonium stock. Course inventory and reference recipes.

sfGFP

Submitted: ilknur_icke_2026-10-06_sfgfp_ribose_mg6975_v1. Selected from the AI-generated wet lab expert example.

Reference: sfGFP Ribose · sfGFP_R2PC_054.

Question: Does reducing magnesium increase functional sfGFP output in the glucose-free ribose/NMP formulation?

Change: Magnesium stock 225 → 175 nL; water 175 → 225 nL. Documented magnesium glutamate decreases from 8.225 to 6.975 mM.

What the previous experiments leave open. Magnesium had already been varied in the RFopt series. A prior 175 nL magnesium condition also contained different potassium, glucose and ribose additions. No exact match for this complete candidate was found in the currently published sfGFP control plate. This is a follow-up in a particular formulation, not a claim that magnesium optimization is new.

Planned comparison. Compare the candidate with the unchanged glucose-free reference on the same experimental run, using matched lysate, geometry and sfGFP maturation time. Include a no-DNA blank and a suitable fluorescence calibration; replicate each condition if the course run permits.

How I would interpret it. Higher calibrated fluorescence would support this lower-magnesium setting in the tested mixture. Lower fluorescence would favor retaining the reference amount. Neither outcome measures free magnesium or identifies whether transcription, translation, folding or maturation caused the difference.

Download the exact submitted JSON.

PETase / TfCut2

Submitted: ilknur_icke_2026-10-06_petase_pep_dsbc2p5_v1. Selected from the AI-generated senior scientist example.

Reference: PETase PEP · Cutinase_R2PC_016.

Question: Can half the supplied DsbC dose retain useful TfCut2 activity?

Change: DsbC stock 1,000 → 500 nL (5 → 2.5 µM); water 1,225 → 1,725 nL. All other additions are unchanged.

What the previous experiments leave open. Every experimental well in the published PETase control plate contained 1,000 nL DsbC. The plate therefore does not answer whether 500 nL is sufficient in this PEP formulation.

Planned comparison. Compare 2.5 µM with the unchanged 5 µM reference in the same run. A later zero-DsbC arm would help establish whether the helper is needed at all. Use matched blanks, the course soluble-ester assay and the same assay handling. Match the DsbC carrier where possible.

How I would interpret it. Similar activity with less DsbC would support reducing that stock input. A substantial activity loss would argue against the cut. The DsbC line item is halved, but total reaction cost is not. Soluble-ester hydrolysis does not establish PET-plastic degradation or a protein mass yield.

Download the exact submitted JSON.

Reteplase

Submitted: ilknur_icke_2026-10-06_reteplase_pep_dsbc3p75_v1. Selected from the AI-generated undergraduate example.

Reference: Reteplase PEP · Reteplase_R2PC_041.

Question: Can a 25% reduction in DsbC retain useful activity while the glutathione mixture is held fixed?

Change: DsbC stock 1,000 → 750 nL (5 → 3.75 µM); water 625 → 875 nL. GSSG remains 4.0375 mM and GSH remains 1 mM.

What the previous experiments leave open. Every experimental well in the published Reteplase control plate retained 1,000 nL DsbC, 475 nL GSSG and 200 nL GSH. Neither reduced-DsbC candidate is present in that dataset.

Planned comparison. Compare 3.75 µM with the unchanged 5 µM reference in the same DSB-compatible lysate and run. Use the course IPR-AMC assay with matched no-DNA background and product calibration. Soluble target measurement and active-enzyme recovery controls would help interpret an activity difference.

How I would interpret it. Retained activity would support this smaller helper dose in the tested conditions. Lost activity could erase any reagent saving. The experiment does not establish therapeutic performance or demonstrate that all nine disulfides formed correctly.

Download the exact submitted JSON.

Cost and output

No protein-output prediction is assigned to these recipes. The sfGFP assay can be calibrated to folded protein concentration; the PETase and Reteplase assays measure product formation or activity, which is not itself a protein mass. Reducing DsbC by 50% or 25% reduces that stock input by the corresponding fraction, not the total reaction cost by that fraction.

The draft guideline prices and live-site cost display are not reconciled. In particular, the live display changed the cost of identical fixed inputs with the selected reference, and PEP/oxalate pricing was unresolved in the local model. I therefore do not report those displays as validated total costs or claim a cost-per-gram improvement. That conclusion requires a consistent cost model and measured functional output.

What I changed in my approach

At first I selected conservative variations of the course references. That made the set easy to describe, but two proposals asked the same broad question about DsbC dose. A small change can still be informative: DsbC was held fixed in the published enzyme screens. I now make the question, comparison and interpretation explicit, and have submitted a fourth design to investigate a different redox question. More ingredients would not automatically mean more learning; one-factor tests can also miss interactions between ingredients.

The prior-test audit covered the currently published control plates: sfGFP_control_20260722-39-L, PETase_control_20260722-39-L, reteplase_control_20260730-DSB, with 312 experimental wells per target. It does not establish novelty across every historical experiment or other students’ proposals. Published course data.

Fourth submitted design · Reteplase glutathione balance

Submitted October 6, 2026: ilknur_icke_2026-10-06_reteplase_pep_glutathione_balance_v1. The course site confirmed this as a separate fourth design; the original three submissions remain in place. No experimental results have been confirmed for this candidate.

Question: Does a less oxidized added glutathione mixture improve Reteplase activity when DsbC and the rest of the PEP reference are held fixed?

The added glutathione-monomer equivalents are equal: [GSH] + 2[GSSG] = 9.075 mM in both mixtures. This holds the number of glutathione units fixed while redistributing reduced and oxidized forms; it does not hold solute-particle concentration fixed or determine actual redox potential. The alternative changes the GSSG and GSH stock additions together as one planned contrast. All other additions match the course reference.

I would compare this alternative directly with the unchanged reference in the same DSB-compatible lysate and run, with matched background, calibration and replication. Higher activity would support the alternative for the tested mixture. Lower activity would suggest that its more reduced input was unfavorable under those conditions. An unchanged result would not prove that redox conditions never matter. Additional soluble-target and assay-interference measurements would be needed to separate effects on protein production, folding, stability and measurement.

Download the fourth submitted JSON · Detailed experiment brief.

B2 · Community reagent: FkpA

I upvoted the existing FkpA (E. coli chaperone) suggestion by Xiangtao Shi on October 6, 2026. The course site confirmed the vote; its button changed to “Remove your vote.”

I chose FkpA to investigate whether folding support beyond DsbC can improve active Reteplase production. Groff and colleagues added purified FkpA alongside DsbC in an E. coli cell-free antibody system and observed improved soluble protein and assembled IgG. That gives a reason to test complementary folding support, but it does not establish a benefit for Reteplase. Groff et al. (2014), Figures 3–4.

The proposed comparison is the unchanged Reteplase reference with and without FkpA, keeping DsbC, glutathione and other ingredients fixed and matching the additive’s carrier. Activity and, if available, soluble and total target protein would be measured. An additive-only assay control would help detect a direct effect on the readout. Increased activity alone would establish a formulation effect, not prove that proline isomerization was the limiting step.

FkpA is a community inventory suggestion. It is not included in any of the four submitted recipes. A usable stock concentration, reagent identifier, price and handling conditions would be needed before making a dispensing recipe.

B3 · Forum access

I can access the cfps-coopetition discussion channel. No bonus optimization post has been sent. One useful question for a later discussion is whether the reference and candidate can be run together with the same lysate batch and assay conditions, so their differences are easier to interpret.

C · Optional plasmid assignments

The optional T7-compatible expression plasmids and helper plasmid are deferred. No plasmid design, construction or laboratory validation is claimed.

AI assistance and prompts

I used Codex (OpenAI) to generate and compare simulated undergraduate, graduate, senior-scientist and wet-lab-expert perspectives, check sources and reaction arithmetic, and prepare this page. The perspective labels describe AI-generated examples, not human contributors. The examples were later revised together using the course inventory. I selected designs from three different examples and then shifted the emphasis toward unanswered experimental questions. Codex carried out the four recipe submissions and the FkpA upvote with my authorization. No new laboratory experiment or measured yield is claimed. Exact model versions were not recorded.

References

• Jewett & Swartz (2004). Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. DOI: 10.1002/bit.20026. Tables I–II and Methods.
• Olsen et al. (2026). Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression. Published paper. Table 1, Figure 2, Methods and Supplementary Tables 1–2.
• Groff et al. (2014). Engineering toward a bacterial “endoplasmic reticulum” for the rapid expression of immunoglobulin proteins. Full text. Figures 3–4; DOI: 10.4161/mabs.28172.
• HTBAA Homework 3 brief, supplied course document, pp. 9–12.
• CFPS coopetition guidelines, supplied draft, stock and price tables and fixed-buffer description.
• Course reaction builder, references and assay descriptions and published control-plate data, checked October 6, 2026.