Week 3 HW: Cell-Free Protocols
A. COMPARE CELL-FREE REACTION COMPOSITIONS
Why does the concentration of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive?
a. Compositions
I believe that magnesium and potassium glutamate differ for different reasons.
Magnesium differs because of the energy substrate where PEP, and the inorganic phosphate it generatesm, combinds and sequesters Mg². Due to such, the PEP based systems is in need of a high total magnesium to keep enough avaliable Mg²⁺ for the ribosomes. Pyruvate binds magnesium weakly and produces almost no phosphate. Because of this, the optimal avaliable magnesium level is reached with even less than half the total magnesium.
Potassium glutamate on the other hand, differs because of ionic/charge balance. As K⁺ and glutamate balance the charge from nucleic acid phosphates, it also recreated a cell alike ionic strength where protein nucleic acid interactions are favored. In the designed system, PEP, HEPES and PEG were removed while acetate was replaced with glutamate salts in addition to the magnesium and ammonium being lowered, it naturally return the ionic environment which mimic the E. coli cytoplasm.
b. Sensitivity
Based on the same reading (Jewett & Swartz) and their optimization studies in both the PANOx-SP (with PEP), I believe that magnesium is more sensitive than potassium. Through data they have presented in their paper (Table II.), magnesium glutamate has performed a sharp peak, a shift 4 mM from the optimum have decreased the harvest to about 62–67% of maximum. Whereas potassium glutamate had a broad optimum where it was able to keep about 95% of maximum yield even when it has increased to 200 mM.
Explain how a PEP+NTP reaction formulation is different from a Ribose+NMP reaction formulation.
I believe that the core difference is how NTP and energy are being supplied to each reaction formulation.
In the PEP+NTP formulation (based on Jewett & Swartz, 2004) the four nucleoside triphosphates (ATP, GTP, CTP and UTP) are being added directly, so RNA polymerase can use them directly. With PEP being a high energy phosphorylated secondary energy substrate, it is regenerating ATP by donating its phosphate to ADP. This reaction formulation is higher in cost as the PEP consumption liberates inhibitory inorganic phosphate and drives the pH down.
In the Ribose+NMP reaction formulation (based on Olsen, 2026) it is cheaper as the nucleoside monophosphates replace NTPs. The lysate’s kinases need to first process through phosphorylate to NTPs before any transcription can continue. ATP in this reaction is regenerated with ribose (added with glucose and phosphate) catabolized by enzymes retained within the lysate, instead of being a donor.
To sum up, they differ in cost (NMP+ribose is cheaper), dependence on metabolism and phosphate (NMP+ribose relies on active lysate metabolism and buffered phosphate), substitutions (e.g. GMP being replaced by guanine + ribose), and reaction kinetics (NMP+ribose is slower and more sensitive to metabolic balance).
Choose two cell-free reaction compositions from the literature, compare and contrast the choice of reagents and the concentration of reagents.
I will be compare and contrasting the cytomim and RFopt reaction. For these two reactions, both of them are glutamate based and PEP free systems that solves the energy concern in opposite ways.
| Reagent | Cytomim (2004) | RFopt (2026) |
|---|---|---|
| Energy / carbon source | Sodium pyruvate 33 mM | Ribose 50 mM + maltodextrin 60 mg/mL |
| High-energy phosphate (PEP) | none | none |
| Nucleotides | NTPs: ATP 1.2, GTP/CTP/UTP 0.85 each | NMPs: AMP 3, CMP/GMP/UMP 2.15 each |
| Phosphate | none added (~4 mM accumulates) | 15 mM added (buffered) |
| Buffer | none (buffer-free) | HEPES 75 mM |
| Potassium glutamate | 130 mM | 362 mM |
| Magnesium glutamate | 8 mM (11.3 total w/ extract) | 8 mM |
| Ammonium glutamate | 10 mM | none |
| Polyamines | Spermidine 1.5 + putrescine 1 | Spermidine 0.13 only |
| Oxalate | 4 mM | none |
| Cofactors | NAD 0.33 + CoA 0.26 (added) | Nicotinamide 4 (NAD precursor); no NAD/CoA |
| tRNA | 170.6 µg/mL (added) | none (relies on lysate) |
| Folinic acid | 0.034 mg/mL | 2.05 mg/mL |
| Amino acids | 2 mM each | 5 mM each |
| Reporter & yield | CAT, ~0.73 g/L (6 h) | sfGFP, 2.4 g/L (15 µL) → 3.7 g/L (bioreactor +O₂) |
Table 1. Reagent comparison between Cytomim and RFopt based on data collected from Jewett & Swartz and Olsen.
Between these two reactions, energy usage is one of the most important differences.
- Cytomim puts pyruvate directly into oxidative phosphorylation where it needs to supply the cofactors that the pathway needs for the reaction such as NAD and CoA. RFopt feeds ribose adding to maltodextrin into glycolysis and respiration. Then, instead of adding NAD directly it uses nicotinamide, letting the lysate rebuild NAD by itself.
- The nucleotides is also different: Cytomim adds triphosphates, while RFopt uses higher concentrated monophosphates (cheaper) and relies on lysate kinases to phosphorylate to the same result.
- Cytomim also eliminates added phosphate and removes the HEPES buffer, whereas RFopt adds buffered phosphate (15 mM) and puts HEPES (75 mM) back in.
- Despite these differences, both independently converged on 8 mM of added magnesium glutamate.
B. DESIGN ONE REACTION COMPOSITION FOR EACH OF THE THREE PROTEINS
This has been designed for for a 20 µL reaction using the reagents from coopetition htgaa.
sfGFP
| Reagent | Unit | Custom Mix | Closest Match |
|---|---|---|---|
| K(Glu) | mM | 220 | 200 (−8.96%) |
| Mg(Glu)₂ | mM | 3.85 | 8.85 (+130%) |
| NH4(Glu) | mM | — | — |
| Ammonium acetate | mM | — | — |
| Amino acids | mM | 5 | 5 |
| Tyrosine | mM | 1 | 1 |
| Cysteine | mM | 1 | 1 |
| HEPES pH 7.5 | mM | 60 | 75 (+25%) |
| Bis-Tris | mM | — | — |
| Phosphate (di:mono) | mM | — | — |
| Phosphate (mono:di) | mM | 15 | 15 |
| Glucose | mM | — | — |
For this design, I have attempted to change components that are related to folding. Such as there are no DsbC, no GSSG/GSH, no catalase as the cysteine stays low. I have also tried to keep it as a simple and plain reducing environment. This design originates from the idea that sfGFP folds on its own with no disulfide bonds. With each oxidizing reagent added it is additional cost that may have little effect on the yield.
PETase
| Reagent | Unit | Custom Mix | Closest Match |
|---|---|---|---|
| K(Glu) | mM | 200 | 200 (0%) |
| Mg(Glu)₂ | mM | 8.85 | 8.85 (0%) |
| NH4(Glu) | mM | — | — |
| Ammonium acetate | mM | — | — |
| Amino acids | mM | 5 | 5 (0%) |
| Tyrosine | mM | 1 | 1 (0%) |
| Cysteine | mM | 1 | 1 (0%) |
| HEPES pH 7.5 | mM | 75 | 75 (0%) |
| Bis-Tris | mM | — | — |
| Phosphate (di:mono) | mM | — | — |
| Phosphate (mono:di) | mM | 15 | 15 (0%) |
| Glucose | mM | — | — |
| Sodium pyruvate | mM | — | — |
In this design, I have added DsbC as the main impact. I believe PETase has a couple of disulfide bonds and it needs support forming and shuffling them correctly. By adding additional DsbC, I wish to provide support and encourage reaction without changing the entire redox environment.
Reteplase
| Reagent | Unit | Custom Mix | Closest Match |
|---|---|---|---|
| K(Glu) | mM | 200 | 200 (0%) |
| Mg(Glu)₂ | mM | 8.85 | 8.85 (0%) |
| NH4(Glu) | mM | — | — |
| Ammonium acetate | mM | — | — |
| Amino acids | mM | 4.81 | 4.81 (0%) |
| Tyrosine | mM | 1.19 | 1.19 (0%) |
| Cysteine | mM | 5 | 5 (0%) |
| HEPES pH 7.5 | mM | 75 | 67.5 (−10%) |
| Bis-Tris | mM | — | — |
| Phosphate (di:mono) | mM | 7.5 | 7.5 (0%) |
| Phosphate (mono:di) | mM | 7.5 | 7.5 (0%) |
| Glucose | mM | — | — |
| Sodium pyruvate | mM | 0.00 | 9.09 |
This design have additional DsbC, GSSG, cysteine and catalase in addition to changes in HEPES. Through the reading, I have learned that GSSG buffer sets an oxidizing environment where many disulfides can form. With Cysteine, it supports the raw thiols where bonds would be built from. Catalase is then used to for oxygen purpose in folding and energy where it produces peroxide that would otherwise damage the reaction. The change in HEPES is then to hold the higher pH where disulfide formation is favored.
HTBAA Upvote I ended upvoting the FkpA (E. coli chaperone) as it provides the most stable structure and yields the most through lower outcome with relatively small changes.