Week 3 HW: Designing a Cell-Free Reaction Composition
CFPS coopetition homework: reaction design for sfGFP, PETase and Reteplase
A. Compare cell-free reaction compositions
A1. Why do potassium glutamate and magnesium glutamate differ between compositions, and which is more sensitive?
The best salt levels are not a fixed property of a recipe. They depend on everything else in the tube, which is why two good formulations can end up with quite different numbers.
Three things move the optimum. The first is the lysate. Extract is made in its own buffer, so it brings Mg and K into the reaction before anything is added. One Swartz-lab description puts this at roughly 3.3 mM Mg and 14.4 mM K in the finished reaction (US 7,338,789, the Jewett & Swartz Cytomim patent), and it shifts with lysate lot and with how much lysate is used. The second is the energy chemistry. Mg2+ is bound by ATP and other nucleotides, by phosphate, and by PEP, so changing the nucleotide or phosphate load changes how much magnesium is actually free. In PEP systems the PEP is partly broken down to inorganic phosphate, and phosphate accumulation from PEP breakdown has been argued to inhibit synthesis, plausibly in part by tying up free magnesium (Jewett & Swartz 2004). Ribose/NMP systems run at a lower phosphate level, around 10 to 15 mM (Olsen et al. 2026). The third is the lysate amount: Olsen et al. had to re-find the best Mg and phosphate for each new lysate batch and for each lysate concentration they tried.
Magnesium is the more sensitive of the two, and there are a few places where you can see it:
- Olsen et al. re-optimized Mg (together with phosphate) for every new lysate batch; they did not describe doing that for K-glutamate.
- In the AI-designed compositions in Smith et al. (2026, Table 2), total Mg(Glu)2 stays inside a narrow 7.0 to 8.8 mM band, while K-glutamate spreads from 273 to 313 mM.
- The same pattern is built into this coopetition’s own six 2026 reference formulations: Mg sits between 8.2 and 10.1 mM in all of them, while K(Glu) ranges from 200 to 345 mM.
- It also shows up in the dispensing arithmetic for our setup: one 25 nL step of the 0.5 M Mg-glutamate stock changes the reaction by 0.625 mM, which matters when the whole optimum is around 8 mM, while one 25 nL step of the 0.875 M K-glutamate stock adds about 1.1 mM on top of the 200 mM that the base buffer already supplies.
The likely reason is that Mg2+ is divalent and binds RNA, ribosomes and nucleotides tightly, so too little starves transcription and translation and too much pushes things toward precipitation and aggregation, while K+ mostly sets ionic strength and is tolerated over a much wider range. That mechanistic explanation is general background, not something I took from one specific paper.
One consequence for my designs: in my first submission (the PEP reference) I left K at the base-buffer 200 mM and only moved Mg. After tabulating the validated ribose/NMP formulations — RF_opt at 362 mM, the coopetition’s ribose references at 273 to 345 mM, the AI top designs at 273 to 313 mM — I changed my mind and added K-glutamate to match each protein’s reference level. K-glutamate costs $0.070/mL, so being wrong costs me about $0.0002 per reaction; being low might cost titer.
A2. How a PEP + NTP formulation differs from a ribose + NMP formulation
In a PEP + NTP system such as PANOx-SP, the lysate is handed finished nucleotides (ATP, GTP, CTP, UTP) and a high-energy phosphate donor, PEP, at around 30 mM. Pyruvate kinase in the lysate moves the phosphate from PEP onto ADP to make ATP again. It is fast — Olsen et al. saw an ATP surge within 30 minutes — but it is wasteful. PEP is also cleaved by phosphatases, so free phosphate builds up (about three times higher than in the ribose/NMP system), and ATP is close to zero by the end of the run. It is also expensive: PEP-based formulations cost upward of $4,000 per liter of reaction in reagents, and Olsen et al. put the phosphorylated energy substrates at over 75% of all material costs.
In a ribose + NMP system (Olsen’s RF_opt), the lysate is handed nucleoside monophosphates (AMP, CMP, GMP, UMP) and a cheap sugar. No NTPs and no phosphorylated energy donor are added. The lysate’s own kinases build the NTPs, and the lysate’s metabolism supplies the energy to phosphorylate them. In Olsen’s data the ATP level reaches a similar peak within 30 minutes without any added ATP and then holds near 250 µM for the whole reaction. Reagents cost about $133 per liter, and the reagent share of total material cost falls from over 75% to under 4%. The trade-offs are that this system depends more on oxygen (a 100% O2 feed raised yield by 45% in their bioreactor, which is part of why 2 mL tubes beat 384-well plates), it is more sensitive to the reaction geometry and lysate amount, and the NMPs only dissolve up to about 5 mM in total (Olsen capped them there to avoid precipitates, and added them last). Smith et al. found that partial replacement of NMPs with cheaper nucleosides or bases works for some nucleotides (AMP with adenosine; GMP with guanine plus ribose) but not for CMP or UMP.
| PEP + NTP (PANOx-SP) | Ribose + NMP (RF_opt) | |
|---|---|---|
| Nucleotide supply | ATP 1.2 mM; GTP/UTP/CTP about 0.85 mM each, added as triphosphates | AMP 3 mM; CMP, GMP, UMP 2.15 mM each, added as monophosphates; lysate kinases make the NTPs |
| Energy source | PEP, about 30 mM, regenerates ATP through pyruvate kinase | Ribose (50 mM) plus the lysate’s own metabolism |
| Phosphate | High, rises as PEP is hydrolysed | About 10 to 15 mM through the run |
| ATP over time | Early surge, near zero by the end | Similar level at 30 min, then held around 250 µM |
| Extra cofactors | NAD, CoA, tRNA, folinic acid, oxalate | Dropped; nicotinamide is used instead (my reading: it is the cheap precursor for the lysate to make NAD) |
| Reagent cost | Upward of $4,000/L ($4,513/L in Olsen Table 1) | $133/L |
A3. Two formulations compared: PANOx-SP (Jewett & Swartz 2004) and RF_opt (Olsen et al. 2026), with the AI-designed set for context
PANOx-SP numbers below are as listed in Olsen et al. (2026) Table 1; note that published PANOx-SP protocols vary between labs (for example Mg-glutamate is reported anywhere from 8 to 16 mM). RF_opt numbers come from Olsen et al. (2026) Table 1, cross-checked against the SOTA column of Smith et al. (2026) Table 2.
| Component | PANOx-SP | RF_opt | AI-designed (Smith 2026, range across six compositions) |
|---|---|---|---|
| Energy | PEP 30 mM | Ribose 50 mM | Ribose 40 to 77 mM |
| Nucleotides | ATP 1.2 mM; GTP, UTP, CTP 0.85 mM each | AMP 3 mM; CMP, GMP, UMP 2.15 mM each | AMP 0.5 to 1.5; CMP 0.4 to 1.1; GMP 0 to 1.1; UMP 0.4 to 1.1 mM |
| Mg glutamate | 8 mM | 8 mM | 7.0 to 8.8 mM |
| K glutamate | 130 mM (plus 10 mM ammonium glutamate) | 362 mM (plus 10 mM ammonium glutamate) | 273.2 to 312.6 mM |
| Buffer | 57 mM HEPES pH 7.5 | 75 mM HEPES-KOH pH 7.5 | 45 to 75 mM HEPES-KOH |
| Phosphate | None added (PEP releases it) | 15 mM potassium phosphate (monobasic:dibasic mix, adjusted to pH 7) | K-phosphate 5.6 to 15 mM; Step 5 data favored 5 to 10 mM |
| Cofactors | NAD 0.33 mM, CoA 0.26 mM, folinic acid 34 µg/mL, tRNA 170.6 µg/mL | Nicotinamide 4 mM | Nicotinamide 0.4 to 4 mM |
| Polyamines | Spermidine 1.5 mM, putrescine 1 mM | None in the Table 2 column | Spermidine 1.2 mM in 3 of 6 designs |
| Amino acids | 2 mM each | 5 mM (plus Tyr 5, Cys 5) | 3.0 to 4.8 mM (Tyr 1.2 to 4.1; Cys 3.2 to 5) |
| Lysate | 30% v/v (as tested by Olsen) | 30% v/v | 25% v/v |
| Performance | Highest yield of the eight older formulations Olsen tested (1.11 ± 0.13 g/L in their hands); $3,760/g, $4,513/L reagents | 2.4 ± 0.3 g/L sfGFP; $133/L reagents; $55/g reagents-only in the preprint (updated to $60/g in the published version, per Smith et al.) | Lowest total cost: $422/g all-in vs $698/g SOTA; best composition 3.04 g/L in 2 mL tubes and 0.72 g/L in 384-well plates (vs 2.39 and 0.25 for SOTA) |
What stands out is how little the first two have in common apart from the salts. PANOx-SP is a give-the-lysate-everything recipe: finished NTPs, a phosphorylated energy donor, three cofactors, tRNA and two polyamines. RF_opt gives the lysate raw materials (NMPs and ribose) and relies on the lysate to build the rest, which removes the expensive items. The salts moved with it: HEPES went up and phosphate was deliberately set, which fits a system that drifts in pH more easily (Olsen et al. saw the pH of RF_opt fall below 7.0 when glutathione was added, and fixed it by using HEPES at pH 7.5).
The AI-designed set kept the ribose/NMP idea and pushed it further: NMPs roughly 2 to 5 times lower than RF_opt with more ribose, adenosine and guanine standing in for part of AMP and GMP, and spermidine added back (their Step 5 median titer was 0.253 g/L with spermidine and 0.131 without). One caution when comparing numbers: Olsen et al. optimized reagent cost only, while Smith et al. optimized total cost per gram including lysate and DNA, which exceed 90% of the cost in the SOTA formulation. So $55 to 60/g (reagents only) and $422/g (all-in) are not the same quantity.
B. My three reaction designs
How I approached the design
Three things drove the choices. First, the cost math. My first submission was essentially the coopetition’s sfGFP PEP reference (a PANOx-SP-style recipe). Priced with the Ginkgo reagent table it comes to $0.0702 per 20 µL reaction (about $3,510/L): PEP alone is $0.0132, the four NTPs together are $0.0188, CoA is $0.0104 and NAD is $0.0025 — about 64% of the reaction cost sits in those six items, and at 2.4 g/L that works out to roughly $1,460/g, two times the $698/g SOTA. That exercise is what convinced me to drop NTPs, CoA, NAD and PEP entirely. In the new designs, lysate (5 µL at $4.00/mL, so $0.020) and DNA (about $0.004) make up about 93% of the reaction cost, and everything else together is under $0.002. So cost per gram is mostly lysate cost divided by titer: cheap reagents that raise titer are close to free, and anything priced like the NTPs is not worth it (1 µL of GTP at $39.34/mL costs $0.039, nearly twice the lysate in the whole reaction).
Second, I started from published ground (RF_opt and the top designs in Smith et al.) and from the coopetition’s own validated 2026 reference formulations, then made changes specific to each protein.
Third, I worked around the fixed parts. The base buffer already supplies 30 mM HEPES-KOH pH 7.5, 200 mM K-glutamate, 2.6 mM Mg-glutamate, 1 mM each of 18 amino acids (including tyrosine) and 1 mM cysteine, and the lysate is fixed at 5 µL, so everything below sits on top of that. All volumes are multiples of 25 nL and, with water, the variable part sums to 11,000 nL (20,000 nL total). Final concentrations are stock × volume / 20,000 nL. I checked every design with a short script: totals exactly 20,000 nL, all steps multiples of 25 nL, total NMP below the 5 mM precipitation cap.
B1. sfGFP (reducing regime)
| Reagent (stock) | Volume (nL) | Final in reaction | Why |
|---|---|---|---|
| HEPES-KOH pH 7.5 (1 M) | 900 | +45 mM (75 mM total) | Buffer; Smith et al. saw median titer 0.177 g/L with added HEPES vs 0.057 g/L without |
| Potassium glutamate (0.875 M) | 3325 | +145.5 mM (345.5 mM total) | Matches the validated sfGFP ribose reference; AI top designs sit at 273 to 313 mM, RF_opt at 362 mM |
| Magnesium glutamate (0.5 M) | 225 | +5.6 mM (8.2 mM total) | Middle of the AI 7.0 to 8.8 mM band; matches all sfGFP references |
| Potassium phosphate 1.6:1 mono:di (0.5 M) | 300 | 7.5 mM | Smith et al.: 5 to 10 mM gave strong titers, none or >25 mM gave almost none |
| Ribose (100 g/L, ~0.67 M) | 1200 | ~40 mM | Lets the lysate build NTPs from NMPs (Olsen et al.); low end of the AI 40 to 77 mM range |
| AMP, CMP, GMP, UMP (0.1 M each) | 200 each (800 total) | 1.0 mM each | NMPs instead of NTPs; 4 mM total, under the 5 mM precipitation cap Olsen et al. used |
| Nicotinamide (0.1 M) | 800 | 4 mM | In RF_opt and 5 of 6 Smith top designs; effectively free ($0.002/mL) |
| Spermidine (0.25 M) | 100 | 1.25 mM | Smith et al.: median titer 0.253 with vs 0.131 without |
| Glucose (200 g/L, ~1.1 M) | 175 | ~9.7 mM | Glucose and phosphate work together (Calhoun & Swartz 2005); glucose appears in 4 of 6 Smith top designs |
| 17 amino acid mix (50 mM each) | 400 | +1 mM each (2 mM total) | See the arithmetic below |
| Tyrosine pH 12 (50 mM) | 200 | +0.5 mM (1.5 mM total) | Same |
| Nuclease-free water | 2575 | n/a | Brings the variable part to 11,000 nL |
The amino acid arithmetic: at about 3 g/L, sfGFP (27 kDa, ~240 residues) is roughly 110 µM, which consumes about 27 mM of amino acid residues. The base buffer supplies about 19 mM of free amino acids (1 mM each of 18 amino acids plus 1 mM cysteine), so it would run short at the titers we are hoping for. The AI-designed compositions run their amino acids higher (3 to 4.8 mM); I stayed lower because the mix is one of the few items with a real volume cost, but this is a knob to scan later.
Estimated cost is $0.0257 per 20 µL reaction (about $1,285/L) including lysate, DNA and base buffer — versus $0.0266 for the coopetition’s own sfGFP ribose reference and $0.0702 for my PEP submission. At $1,285/L I need about 1.8 g/L to match the $698/g SOTA and about 3.0 g/L to match the $422/g that Smith’s best composition reached in tubes. Plate geometry will likely give lower titers than tubes (Smith et al. saw 0.72 vs 3.04 g/L for their most balanced composition), so I expect the 384-well numbers to look worse than the tube numbers.
B2. PETase / TfCut2 (reducing regime, one disulfide)
TfCut2 sits in the same DTT-containing regime as sfGFP, so I kept the same chassis and changed a few things on purpose.
| Reagent (stock) | Volume (nL) | Final in reaction | Why |
|---|---|---|---|
| HEPES-KOH pH 7.5 (1 M) | 900 | +45 mM (75 mM total) | As for sfGFP |
| Potassium glutamate (0.875 M) | 2275 | +99.5 mM (299.5 mM total) | Matches the PETase ribose reference |
| Magnesium glutamate (0.5 M) | 225 | +5.6 mM (8.2 mM total) | As for sfGFP |
| Potassium phosphate 1.6:1 mono:di (0.5 M) | 300 | 7.5 mM | As for sfGFP |
| Ribose (100 g/L) | 1200 | ~40 mM | As for sfGFP |
| AMP, CMP, GMP, UMP (0.1 M each) | 200 each | 1.0 mM each | As for sfGFP (4 mM NMP total) |
| Nicotinamide (0.1 M) | 800 | 4 mM | As for sfGFP |
| Spermidine (0.25 M) | 100 | 1.25 mM | As for sfGFP |
| Glucose (200 g/L) | 175 | ~9.7 mM | As for sfGFP |
| 17 amino acid mix (50 mM each) | 500 | +1.25 mM each (2.25 mM total) | TfCut2 is slightly larger than sfGFP (~260 vs ~240 residues in the active enzyme) |
| Tyrosine pH 12 (50 mM) | 200 | +0.5 mM | Same |
| Oxidized glutathione (0.17 M) | 75 | 0.64 mM | Low-dose oxidant as a bet for the single disulfide |
| Nuclease-free water | 3450 | n/a |
Two honest notes on this design. First, the GSSG is the part I am least sure about. The standard lysate carries DTT, so the glutathione may simply be reduced and do nothing, and I would not be surprised by that. It costs about $0.00005 per reaction, so it is a cheap test of whether a gentle redox nudge helps the one disulfide form. Second, the coopetition’s own PETase reference formulations include 5 µM DsbC — at $5,632/mL that is $5.63 per reaction, more than 200 times everything else in the reaction combined, so I deliberately left it out. If PETase activity comes back poor, low-dose DsbC (0.25 to 0.5 µM) is my first fallback experiment.
Estimated cost is $0.0260 per reaction (about $1,300/L). Because the PETase assay is activity-based and measured after a 10 minute 60 °C step that should knock out the lysate’s own esterases, an enzyme that is stable at 60 °C matters — which is part of why I propose calcium in B5.
B3. Reteplase (oxidizing / DSB regime)
Reteplase has nine disulfide bonds, so it needs the oxidizing setup described in the Ginkgo overview: DSB lysate (no DTT, iodoacetamide-treated, cysteine-quenched), a glutathione redox pair, and DsbC. One practical note: the guidelines say Ginkgo prepares the DSB lysate in separate reagent plates “for future use in reteplase CFPS reactions”, so I read the fixed 5,000 nL lysate slot as being filled with DSB lysate automatically for reteplase plates — I will confirm on the submission page whether the JSON takes the standard lysate field or a separate DSB lysate entry.
| Reagent (stock) | Volume (nL) | Final in reaction | Why |
|---|---|---|---|
| HEPES-KOH pH 7.5 (1 M) | 1200 | +60 mM (90 mM total) | Olsen et al. found glutathione drops the pH of a ribose/NMP reaction below 7.0; their fix was HEPES at pH 7.5 (which our stock already is), and Smith et al. saw added HEPES raise titer, so I stay at the top of the range |
| Potassium glutamate (0.875 M) | 1675 | +73.3 mM (273.3 mM total) | Matches the reteplase ribose reference |
| Magnesium glutamate (0.5 M) | 250 | +6.25 mM (8.85 mM total) | Matches the reteplase ribose reference |
| Potassium phosphate 1.6:1 di:mono (0.5 M) | 300 | 7.5 mM | Dibasic-rich mix, against the acidification risk |
| Ribose (100 g/L) | 1200 | ~40 mM | As for the other two |
| AMP, CMP, GMP, UMP (0.1 M each) | 200 each | 1.0 mM each | 4 mM NMP total, under the 5 mM cap |
| Nicotinamide (0.1 M) | 800 | 4 mM | As for the other two |
| Spermidine (0.25 M) | 100 | 1.25 mM | As for the other two; Yin & Swartz also found polyamines helpful for disulfide proteins |
| 17 amino acid mix (50 mM each) | 500 | +1.25 mM each | Large protein (355 aa, ~39 kDa) |
| Tyrosine pH 12 (50 mM) | 200 | +0.5 mM | Same |
| Cysteine (0.2 M) | 200 | +2 mM (3 mM total) | Reteplase has 18 cysteines per molecule; at ~80 µM product that is ~1.4 mM of cysteine consumed, more than the 1 mM the base buffer gives. The reference uses 5 mM total; I went lower to keep the redox tilted oxidizing |
| Oxidized glutathione (0.17 M) | 475 | 4.04 mM | Oxidizing environment |
| Reduced glutathione (0.1 M) | 200 | 1.0 mM | 4:1 GSSG:GSH, the ratio used by Yin & Swartz (2004), Kim & Swartz (2003) and Olsen et al. for disulfide proteins |
| DsbC (100 µM) | 100 | 0.5 µM | Isomerase for the nine disulfides; see cost note |
| Nuclease-free water | 3000 | n/a |
A few deliberate omissions. I left glucose out because glucose metabolism can acidify the reaction and I am already working against a pH drop from glutathione — that is my own reasoning rather than something I found tested. I also left out the sodium pyruvate, adenosine and catalase that appear in the reteplase ribose reference (it is essentially Smith’s composition 1793122_35 plus the DSB components); those are my round-two tests rather than part of the base design.
The DsbC dose is the whole cost story for this protein. At $5,632/mL, 100 nL of DsbC is $0.563 per reaction — about 95% of the roughly $0.59 total and 28 times the lysate cost. The reference formulations use 5 µM, ten times my dose, which would be $5.63 per reaction (about $283,000/L); Yin & Swartz (2004) used 75 µg/mL, about 3 µM. DsbC is catalytic (it shuffles disulfides rather than being consumed stoichiometrically), so a sub-micromolar dose is plausible, but this is a genuine risk and the dose-response in B4 is my highest-value experiment for this protein. Reteplase plates also run only 10 hours at 30 °C before the cold and ambient holds, against 20 hours for sfGFP and PETase, so the ribose/NMP system’s fast ATP start (within about 30 minutes in Olsen et al.) is useful here.
Estimated cost is $0.590 per reaction (about $29,500/L). For scale: if active reteplase comes out at the ~60 µg/mL that Yin & Swartz reached for vtPA, that is about $490,000/g at my 0.5 µM DsbC — and about $4.7 million/g at the reference’s 5 µM. Folding yield and the DsbC dose are basically the whole game for this protein.
(Key names above follow the export format of my first submission JSON; I will double-check the exact keys for the phosphate mixes, glutathione and DsbC against the submission form, which validates them automatically.)
B4. What I would test next (draft experiments)
Each 384-well plate holds 80 unique compositions in quadruplicate, so each plan below fits on one plate with room to spare.
sfGFP plate — Mg × phosphate grid plus one-factor tweaks. The two settings Olsen et al. had to re-tune for every lysate are Mg and phosphate, so: total Mg at 6.5, 8.2, 10 and 12 mM (150, 225, 300, 375 nL of the 0.5 M stock) crossed with phosphate at 0, 5, 7.5 and 10 mM (0, 200, 300, 400 nL) on my B1 chassis = 16 designs. Then one-factor rows on the best cell: K-glutamate at 200 / 280 / 345 mM total; spermidine 0 / 1.25 mM; NMP total 2 / 4 / 5 mM; glucose 0 / 9.7 mM; ribose 40 / 50 / 77 mM. Readout is direct fluorescence, so this plate also calibrates how far the plate format is from tubes.
PETase plate — redox and DsbC. My B2 chassis with: GSSG at 0 / 0.64 / 2 mM; DsbC at 0 / 0.25 / 0.5 µM (kept low on purpose for cost); and the reference PETase ribose composition as a positive benchmark. If calcium chloride gets added to the inventory (B5), repeat the best condition with CaCl2 at 0.5 / 1 / 2 mM and check whether the 60 °C assay step leaves more active enzyme.
Reteplase plate — DsbC dose-response crossed with redox ratio. DsbC at 0, 0.25, 0.5, 1, 2.5 and 5 µM crossed with GSSG:GSH at 4:1 (4.04:1 mM), 2:1 and 8:1 = 18 designs, plus added-cysteine at 0 / 2 / 4 mM on the best cell, plus HEPES at 75 vs 90 mM total. The DsbC row alone spans $0 to $5.63 per reaction, so this plate directly measures the price of folding.
B5. Community reagent proposal: calcium chloride
My proposal is calcium chloride as a 100 mM aqueous stock (calcium acetate would be a fine alternative). TfCut2 has a calcium binding site, and adding Ca2+ or Mg2+ raised the thermostability of the Thermobifida fusca polyester hydrolases, enabling PET degradation closer to the glass transition temperature (Then et al. 2015); the calcium binding site residues in TfCut2 are characterized structurally (Roth et al. 2014, PDB 4CG1), and engineering a disulfide into that calcium site made the enzyme calcium-independent and more thermostable, with wild-type TfCut2 melting at about 70 °C (Then et al. 2016). Our assay heats the reaction to 60 °C for 10 minutes, which TfCut2 should survive, but not by a huge margin, so extra stability could protect the signal. Mg is already on the menu, but Mg is dictated by what translation needs, so a separate calcium knob would let us stabilize the enzyme without moving the Mg optimum.
How I picked it: I went through the menu and saw that Mg is the only divalent metal, read the TfCut2 structure and stability papers (Roth et al. 2014; Then et al. 2015; Then et al. 2016), and chose the one additive with direct evidence for this exact enzyme that is also cheap and easy to buy. The main risk is that calcium can precipitate with phosphate and nucleotides, so I would test 0.5 to 2 mM and keep phosphate at or below 7.5 mM. I have not seen data on how calcium affects translation in this lysate. [Add: I submitted / upvoted this on the Community Reagents page on (date).]
I also upvoted Skp (already proposed by another team): Yin & Swartz (2004) found that 300 µg/mL Skp raised the solubility of cell-free-made vtPA (reteplase) by 60 to 70% and brought active yield to about 60 µg/mL, while PDI, DsbA, GroEL/GroES and DnaK/DnaJ/GrpE gave no significant benefit in the same system.
Links: Then et al. 2015, https://doi.org/10.1002/biot.201400620 ; Then et al. 2016, https://doi.org/10.1002/2211-5463.12053 ; Roth et al. 2014, https://doi.org/10.1007/s00253-014-5672-0 ; Yin & Swartz 2004, https://doi.org/10.1002/bit.10827
Bonus idea posted to the Zulip channel
DsbC is about 95% of my reteplase reaction cost, so a dose-response across plates (0 to 5 µM) is probably the highest-value experiment for that protein. It may also be worth asking whether a cheaper source of isomerase activity (for example lysate from a DsbC-overexpressing strain, or in-house purified DsbC) could be added to the reagent inventory.
Use of AI
I used Claude (Anthropic) to break down the Ginkgo coopetition document and the reference papers, create figures and help do some mathematical calculations.
References
- Smith, A. A., et al. (2026). Using a GPT-5-driven autonomous lab to optimize the cost and titer of cell-free protein synthesis (Ginkgo Bioworks × OpenAI). bioRxiv. https://www.biorxiv.org/content/10.64898/2026.02.05.703998v1
- Olsen, M. L., et al. (2026). Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression. Nature Communications. https://doi.org/10.1038/s41467-026-69605-8 (preprint: https://www.biorxiv.org/content/10.1101/2025.08.01.668204v1)
- Jewett, M. C., & Swartz, J. R. (2004). Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnology and Bioengineering, 86(1), 19–26. https://doi.org/10.1002/bit.20026
- Jewett, M. C., & Swartz, J. R. (2008). Methods of in vitro protein synthesis. US Patent 7,338,789 B2.
- Calhoun, K. A., & Swartz, J. R. (2005). Energizing cell-free protein synthesis with glucose metabolism. Biotechnology and Bioengineering, 90(5), 606–613.
- Kim, D.-M., & Swartz, J. R. (2003). Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnology and Bioengineering, 81(2). https://doi.org/10.1002/bit.10865
- Yin, G., & Swartz, J. R. (2004). Enhancing multiple disulfide bonded protein folding in a cell-free system. Biotechnology and Bioengineering, 86(2), 188–195. https://doi.org/10.1002/bit.10827
- Then, J., et al. (2015). Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fusca. Biotechnology Journal, 10(4), 592–598. https://doi.org/10.1002/biot.201400620
- Then, J., et al. (2016). A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate. FEBS Open Bio, 6(5). https://doi.org/10.1002/2211-5463.12053
- Roth, C., et al. (2014). Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca. Applied Microbiology and Biotechnology, 98. https://doi.org/10.1007/s00253-014-5672-0
- Silverman, A. D., Karim, A. S., & Jewett, M. C. (2020). Cell-free gene expression: an expanded repertoire of applications. Nature Reviews Genetics, 21, 151–170. https://doi.org/10.1038/s41576-019-0186-3
- Rasor, B. J., et al. (2021). Toward sustainable, cell-free biomanufacturing. Current Opinion in Biotechnology, 69, 136–144. https://doi.org/10.1016/j.copbio.2020.12.012
- Ginkgo Bioworks CFPS coopetition: overview, guidelines and reagent table (course handout); plate model and validator: https://github.com/ginkgobioworks/autonomous-cfps