Autonomous biogenesis of all thirty proteins of the Escherichia coli translation machinery

Domain: physics
Published: 2025-12-30
Editorial outcome: minor_revision

Abstract
The cell-free biogenesis of the protein translation machinery is essential for the creation of a self-regenerating synthetic cell. Here, we demonstrate the autonomous and simultaneous biogenesis of all thirty proteins of the translation machinery of E. coli in a reconstituted transcription-translation system. We first establish self-regeneration of every translation protein by determining the threshold concentration required for its own synthesis from a synthetic gene coding for the protein, thereby demonstrating the functionality of all nascent proteins, separately. Simultaneous biogenesis of multiple translation proteins at their threshold results in delayed synthesis below detection levels. To achieve self-regeneration of multiple translation proteins, we induce boundary-free compartmentalization of the reaction by immobilizing the genes on a surface at high density. The co-localization of genes, molecular machinery, messenger RNA and nascent proteins at the surface creates sufficient conditions to catalyze the simultaneous self-regeneration of sub-groups up to all thirty translation proteins, as measured by total internal reflection fluorescence on the surface. Our approach provides mechanistic insight and presents a general methodology for the biogenesis of cellular machines toward autonomous synthetic systems.

Introduction
Recreating the conditions for autonomous biogenesis of the protein translation machinery is fundamental to our understanding of living systems and is essential for the establishment of minimal cell models1. A major step forward was the cell-free reconstitution of genetically encoded protein synthesis, defining the minimal set of purified protein factors and enzymes that support multiple translation cycles2 (Fig. 1a). This cell-free bacterial platform, termed the PURE system, has been shown throughout the years to support the synthesis of a myriad of proteins3–8. Finding conditions that enable the minimal translation system to exhibit biogenesis and self-regeneration is the next essential milestone toward an autonomous protein-based artificial cell9,10. We focus on the development of a general approach for the autonomous co-synthesis of thirty protein factors and enzymes, including twenty aminoacyl transfer RNA (tRNA) synthetases (aaRS) and ten translation factors (Fig. 1a), that allows for the nascent proteins to actively integrate back into the system that created them (Fig. 1b).

Intuitively, the demand for the co-synthesis of thirty different proteins at sufficiently high concentrations calls for a reconstituted system that operates at the highest possible capacity. While improved versions of the PURE system with increased protein yields have been established11–13, they contain high concentrations of the initial machinery. Therefore, the contribution of newly synthesized translation factors and enzymes would have little observable effect when the system operates at saturating synthesis rates (Fig. 1c, right panel). This challenge could be resolved by diluting the reaction after sufficient proteins had been synthesized, to allow identification of the nascent components. Using this approach, the self-regeneration of T7 RNA polymerase, two kinases and RF1 and 12 aaRS was demonstrated using a serial dilution experiment of two cycles14. In a separate study, all 20 aaRS were simultaneously self-regenerated from their corresponding genes in a serial dilution series of up to 20 cycles15. Furthermore, the self-regeneration of up to seven aaRS was achieved within a PURE system diluted using a microfluidic device16.

Here, we demonstrate an alternative autonomous approach, starting with a system that operates at its threshold level under extremely diluted conditions of initial PURE translation proteins. In this scenario, functional nascent translation proteins are synthesized from their genes and accumulate very slowly, feeding back into the system until a threshold concentration is crossed, producing a significant rise in activity due to self-amplification (Fig. 1c, left panel). For such a scenario to succeed, a mechanism to catalyze and enhance the reaction at low machinery concentrations during the initial stages is required. Recently, we presented a cell-free scenario for the localization of gene expression reactions at surface-immobilized high-density gene brushes as boundary-free compartments17,18, leading to the accumulation of nascent RNA and protein products and increasing their local concentration to facilitate macromolecular interactions. We demonstrated co-translation assembly of T4 bacteriophage structural modules and reconstituted the biogenesis of functional E. coli RNA polymerase19. In addition, by encoding 26 different ribosomal proteins and assembly factors and one scaffold ribosomal RNA in such localized reaction centers, we were able to demonstrate the biogenesis, hierarchical order, and assembly timeline of the E. coli small ribosomal subunit20. Here, we utilize this approach, immobilizing all the genes coding for the thirty protein factors and enzymes of the translation machinery in a DNA brush to demonstrate their autonomous co-synthesis and functional integration back into the machinery that created them. Other scenarios to confine and co-localize the reaction components, for example, by packing multiple genes as continuous synthetic genomes in enclosed compartments, could also be envisioned using the same general principles21–24.

As part of our general self-regeneration strategy, we first determine in bulk solution reactions for each translation protein (Pi) separately the threshold concentration below which the synthesis rate of a reporter green fluorescence protein (GFP) is strongly reduced. Upon addition of the genes coding for the corresponding translation protein, we detect an increase in the reporter signal due to crossing the threshold concentration by self-synthesis. When self-regeneration for individual or multiple proteins can not be demonstrated in a bulk solution, we adopt the approach of localized reaction centers. We immobilize the corresponding genes as dense DNA brushes, add the dilute PURE reaction mixture with each protein at its threshold concentration, and use total internal reflection fluorescence (TIRF) microscopy for sensitive detection of a surface-trapped GFP reporter. At the threshold level, the minimal system is unable to support GFP synthesis unless all genes coding for the translation proteins are included in the DNA brush, allowing us to define the transition point to cell-free biogenesis of all thirty translation proteins simultaneously from the bottom up.

Results
A self-regeneration assay for the protein translation machinery
The thirty protein factors and enzymes that are essential for in vitro protein translation belong to four biochemically distinct subgroups: twenty aaRSs, each of which covalently charges a tRNA with its cognate amino acid; and four initiation, three elongation and three termination factors which together comprise a total of ten proteins involved in the three stages of the translation cycle (Fig. 1a). We introduced all thirty translation genes and the GFP reporter gene to a PURE bulk reaction (Fig. 1b) and monitored the fluorescence intensity time trace, FI(t), against control reaction with dummy genes coding for unrelated protein of a similar synthesis load as the translation proteins (Fig. 1d). Since there was no significant difference between the two fluorescence time traces, we were unable to deduce the synthesis and functionality of the nascent translation machinery. We verified that each of the translation machinery proteins was synthesized from its gene in the minimal protein translation system (Supplementary Fig. 1). Therefore, the functionality of the nascent translation proteins may have been masked because the initial PURE reaction was already saturated at the reaction conditions (Fig. 1c).

We reasoned that a very dilute PURE mix in which all the translation machinery proteins were at a very low initial concentration would be a more suitable scenario for self-regeneration, as it allows for a bottom-up buildup of all components and provides a definitive manifestation of the activity of nascent machinery (Fig. 1c). To support this scenario, we performed numerical simulations of protein synthesis in the PURE system25 with different initial concentrations of all thirty translation factors (Supplementary Fig. 2) and found a dilution regime where the translation rate is more sensitive to protein concentrations than in a full PURE. We then simulated the biogenesis of one of the translation proteins (P), asparaginyl-tRNA synthetase (AsnRS), for two different scenarios: (i) with biogenesis, that is, the nascent AsnRS proteins are active and feedback into the pool of initial AsnRS, or (ii) without biogenesis, that is, the nascent proteins are synthesized but are inactive and just accumulate (Fig. 1e). Consistent with autocatalytic biogenesis, once nascent AsnRS exceeded a threshold, its maximum synthesis rate increased sharply compared with non-autocatalytic production. For higher initial concentration of AsnRS, the translation rates at both scenarios converged and plateaued, coinciding with the saturating regime of the full PURE reaction (Fig. 1d and Supplementary Fig. 2).

Empirical determination of threshold conditions for self-regeneration
Based on the simulation, we sought to determine empirically the threshold concentration of each Pi in an initial PURE composition at which GFP fluorescence could be barely detected. The addition of genes coding for this Pi should result in its synthesis and accumulation to a concentration sufficiently above the threshold to produce GFP, indicating self-regeneration. We first set 30 parallel PURE reactions, each with one of the proteins removed completely (ΔPi), expecting to obtain no GFP synthesis since each Pi component should be essential for PURE activity2. For some ΔPi reactions, we obtained negligible GFP signals as expected, but for others (i.e., for all the initiation and termination factors and some aaRSs), there were substantial GFP signals that most likely originated from Pi contamination. This background protein expression level was highly dependent on the source of the added ribosomes suggesting they were the main source of contamination (Supplementary Fig. 3 and 4). Mass spectrometry analysis demonstrated directly that the ribosome preparations indeed included translation machinery proteins (Supplementary Fig. 5). The commercial preparation of ribosomes (see “Methods”) had the overall lowest residual activity for most of the ΔPi, excluding the initiation and release factors, which had substantial residual activity throughout all ribosome preparations (Supplementary Fig. 4). To minimize masking of the self-regeneration activity, we therefore chose to use the commercial preparation of ribosomes in the rest of the study. In one batch reaction, our full PURE mix with the commercial ribosomes produced around 1600 nM of GFP with a max. rate of 14 nM min−1 (Supplementary Fig. 6).

We then introduced the gene coding for Pi at different concentrations in ΔPi-PURE and monitored the GFP expression rate. If no increase was observed, we titrated purified Pi back into the ΔPi-PURE mix to determine the threshold and dynamic range for a GFP response and then reintroduced the gene at that threshold concentration. For example, at an initial Valyl-tRNA synthetase (ValRS) concentration of 1 nM, the addition of the ValRS gene resulted in a significant boost in the synthesis rate of the reporter protein compared to the reaction without the added ValRS gene, indicating that the nascent ValRS was functional and integrated back into the PURE machinery (Fig. 1f, g and Supplementary Fig. 7). This analysis allowed us to observe ValRS self-regeneration for initial concentrations between 0.25 to 2.5 nM (Fig. 1f, g). Notably, we detected an inflection point in the GFP synthesis rate at about 20 to 30 min, even without the aaRS genes, indicating low rates of protein synthesis in the initial PURE reaction (Fig. 1f). In the presence of aaRS genes, a second peak appeared after a significant delay (Fig. 1f and Supplementary Fig. 7), which we attribute to the activity of nascent ValRS contributing both to GFP and its own synthesis. The time of the second peak (tmax) decreased as the initial ValRS concentration increased (Fig. 1h and Supplementary Fig. 7).

We repeated this analysis for the other twenty-nine translation proteins and summarized the effect of supplementing the gene on the protein synthesis rate at the threshold concentration in Fig. 1i. For each of the twenty aaRS we found threshold conditions that establish their self-synthesis and functionality, similarly to the ValRS analysis. For ten of the aaRSs (Arg, Asn, Cys, Glu, His, Lys, Met, Thr, Trp, and Tyr), the residual activity in a ΔPi-PURE was enough to kick-start the self-synthesis upon addition of the corresponding gene (Fig. 1h and Supplementary Fig. 8), resulting in increased maximal GFP synthesis rate (max. rate), and implying that the nascent aaRS was functional. Beyond a certain gene concentration, often around 1 nM, the max. rate decreased again, which could be attributed to a “competition” effect, where increasing the DNA of one gene reduces the expression of another gene (GFP), most likely due to the sharing of resources26 (Supplementary Fig. 9). Specifically, for ArgRS, we found that its synthesis required the addition of a protein chaperone mix (see Methods) which we thus added to all reactions (Supplementary Fig. 10). For the other ten aaRSs (Ala, Asp, Gln, Gly, Ile, Leu, Phe, Pro, Ser, and Val) (Fig. 1h and Supplementary Fig. 11), the threshold concentration was found to be, on average, between 1 and 2% of the concentration of the aaRS in a full reaction, often reaching sub-nanomolar concentration, with GlyRS being the exception, requiring 42% of its full reaction concentration (Supplementary Table 1).

For the three elongation factors, we recalled that their concentration in a full PURE reaction is at the micromolar range. Accordingly, the threshold concentration and dynamic range of two elongation factors, EF-G and EF-Ts, were found to be about ten times higher compared to the threshold concentration of the aaRSs (Supplementary Figs. 12 and 13). Still, we could demonstrate their self-regeneration upon addition of their respective genes, observing an increase in the yield and the max. rate and a decrease in the tmax (Fig. 1i and Supplementary Figs. 12 and 13). For EF-Tu, we could not differentiate between the functionality of initial or nascent EF-Tu, most likely due to the broad dynamic range of the GFP response function to EF-Tu concentration, from a few nanomolar to several micromolar (Fig. 1i and Supplementary Fig. 14). In addition to EF-Tu, we could not demonstrate at this stage self-regeneration of some of the translation initiation and termination factors (IF1, IF3, MTF, RF1, and RF3), as individual removal of each from the reaction did not significantly reduce the protein synthesis rate (Supplementary Fig. 4). We only observed some indication for self-regeneration for IF2 and RRF (Supplementary Fig. 15).

Self-regeneration of groups of initiation and release factors
The self-regeneration in bulk solution for the initiation and termination factors in corresponding ΔPURE versions was masked by substantial contaminations far beyond threshold conditions (Supplementary Figs. 4 and 5). We thus hypothesized that we could create threshold conditions by removing multiple factors simultaneously from the initial PURE reaction (Fig. 2a). Indeed, the max. rate dropped for the simultaneous deletions of all four initiation factors (IF1+2+3+MTF) or for the mutual deletion of the release factors (RF1+RF3+RRF) and was restored in the presence of their respective genes (Fig. 2b). By using permutations of the four- and three-member initiation (IF1, IF2, IF3, and MTF) and release groups (RF1, RF3, and RRF), respectively, we were able to dissect the contribution of each protein (Fig. 2c, d and Supplementary Fig. 16). The difference in max rates with and without their respective genes (Δkmax) showed an increase for all combinations except for Δ(IF1+MTF) and Δ(RF1+RF3), but their functionality could be deduced from the rest of the combinations. Interestingly, in combining the initiation factors from one to four, we observed an extension in tmax, from a few min to 40 min (Fig. 2c). We did not observe such extension in tmax as more release factors were deleted, but the Δkmax for Δ(RF1+RF3) increased slightly compared to the single deletions (Fig. 2d), and the other group deletions were dominated by RRF contribution. Despite substantial contaminations of these protein factors from the ribosomes in the initial reaction, we could conclude from the bulk solution reactions that the seven nascent initiation and release factors were participating in GFP synthesis upon their self-synthesis.

Self-regeneration of EF-Tu at surface-localized reaction centers
Demonstrating self-regeneration of EF-Tu was challenging, and we therefore sought evidence that nascent EF-Tu synthesized in the PURE was functional by separating the reaction into two consecutive steps (Fig. 3a) (see Methods). In the first step, we added the EF-Tu gene to a PURE reaction with lowered EF-Tu concentration, and after incubation, allowing for sufficient EF-Tu synthesis to occur (reaction 1), we transferred a small volume fraction to a fresh ΔEF-Tu-PURE reaction supplemented with GFP genes (reaction 2). We observed that the GFP signal in reaction 2 increased with the EF-Tu gene concentration in reaction 1, thus confirming that nascent EF-Tu synthesized in a PURE reaction was functional (Fig. 3b, c and Supplementary Fig. 17). Self-regeneration of EF-Tu was most likely hidden by the high threshold concentrations necessary for efficient protein synthesis in a one-step coupled system.

To increase the efficiency of EF-Tu self-regeneration at reduced threshold concentrations, we created localized reaction centers of EF-Tu genes immobilized on a surface as high-density DNA brushes within a large reservoir of nutrients (Fig. 3d). This design served two purposes: first, to overcome the capacity barrier imposed by the limited nutrient supply and reduced EF-Tu concentration; and second, to spatially co-localize EF-Tu synthesis to the site of GFP synthesis, where EF-Tu activity is required. The genes were arranged in clusters of three identical DNA brushes (Fig. 3d, e). Within each cluster the brushes contained a different fraction of EF-Tu genes, from 0 to 80%, and in addition, a 2% gene fraction coding for GFP fused to a Hemagglutinin tag (GFP-HA). The surface surrounding the DNA brushes was patterned with HA-specific antibodies for trapping nascent GFP-HA on the surface for sensitive detection by TIRF imaging (Fig. 3d–f). The overall DNA density and, thus, gene expression rate were kept constant by supplementing brushes with DNA coding for an unrelated (dummy) gene17 (see Methods). Upon addition of a PURE reaction mixture initially containing 1 µM purified EF-Tu, we observed, over the course of 1–2 hours, accumulation of GFP-HA on the surface in the vicinity of the DNA brushes. This demonstrates, in a single reaction, that the nascent EF-Tu synthesized from the brushes is functional in the PURE system and essential for GFP-HA synthesis (Fig. 3g–i, Supplementary Fig. 18 and Supplementary Movie 1). The GFP-HA signal increased in response to larger fractions of EF-Tu genes up to 40%, with a slight decrease at 80% most likely due to resource competition or inhibiting byproducts.

Simultaneous self-regeneration of sub-groups of translation machinery proteins
Encouraged by the demonstration of self-regeneration of EF-Tu when encoded in DNA brushes, we attempted simultaneous biogenesis of all three elongation factors encoded in the same DNA brushes, with the ratio of the respective genes set by the results from the single self-regeneration assays (Supplementary Table 3). Upon addition of a translation reaction with purified EF-Ts, EF-G, and EF-Tu set at threshold concentrations, GFP-HA signals accumulated on the surface surrounding DNA brushes in response to the increase of EFs genes fraction, with a maximum GFP signal at 40% and a sharp drop at 80% (Fig. 3j–l, Supplementary Fig. 18 and Supplementary Movie 2). We concluded that the localization of the translation machinery at the DNA brushes co-localized the synthesis of all the nascent elongation factors in the vicinity of the brushes at sufficient concentrations to self-amplify their own synthesis as well as GFP synthesis18.

Towards self-regeneration of all twenty aaRSs simultaneously, we recalled that they comprise 20 distinct enzymes (Fig. 4a). This diversity motivated us to investigate whether specific parameters within this group might influence the self-regeneration process. To this end, we compared the max. rate in the self-regeneration assays (Fig. 1 and Supplementary Fig. 8) with respect to various physical and biochemical parameters—including kinetic rate constants, number of cognate amino acids, protein or substrate amino acid size and amino acid hydrophobicity27 (Supplementary Figs. 19 and 20). We looked for correlations between physical parameters of the aaRS and Δkmax, the difference between the GFP max. synthesis rate with and without the aaRS gene, and found a moderate negative correlation between Δkmax and the length of the aaRS genes as well as the hydrophobicity of the substrate amino acid, and moderate positive correlation between Δkmax and the gel band intensity for its own expression (Fig. 4b and Supplementary Fig. 20). We did not observe a correlation between Δkmax and the number of amino acids in the reporter protein. The only GFP sequence effect we may have observed was for the ΔTrpRS-PURE reactions, where residual GFP production was higher than for other aaRS deletions. Since GFP contains only a single Trp, the residual GFP signal might be attributed to misincorporation at this position. However, adding the TrpRS gene still boosted the rate substantially, supporting the conclusion that TrpRS could be self-regenerated. We were encouraged that there was no apparent obstacle to simultaneous self-regeneration of all twenty aaRSs, except for their load on the PURE reaction. Indeed, when attempting co-synthesis of all twenty aaRSs in bulk reaction, the GFP signal was reduced compared to a bulk reaction without the aaRSs genes (Fig. 4c and Supplementary Fig. 21). The combined synthesis yield of all the twenty aaRSs was most likely not sufficient to cross the threshold concentrations required to demonstrate self-biogenesis.

Inspired by the biogenesis of the elongation factors in the localized-reaction centers, we created DNA brushes that contain different fractions of all the twenty genes of the aaRS, and a 2% constant GFP-HA fraction (Fig. 4d). We added a PURE reaction with ten aaRSs completely removed and ten at their threshold concentrations. We observed GFP accumulation in the proximity of the DNA brushes dependent on the amount of aaRS genes in the brush (Fig. 4e, f; Supplementary Fig. 22 and Supplementary Movie 3). We concluded that co-localizing the synthesis of all the nascent aaRSs in the vicinity of the DNA brushes yielded sufficient apparent concentrations to support self and GFP synthesis, thus demonstrating simultaneous biogenesis of twenty aaRSs in one minimal translation reaction, which was not possible in a bulk solution.

Direct observation of nascent protein localization to gene brushes
To gain mechanistic insight into the biogenesis of the translation machinery catalyzed by DNA brushes, we created C-terminal GFP fusions of several translation proteins (see Supplementary Data File 1). The transient accumulation of nascent protein fusions at the vicinity of their DNA brushes could be detected by the localized TIRF illumination of the surface, implying their participation in gene expression18 (Fig. 5c). We first tested the functionality of C-terminal GFP fusions in bulk solution reactions using mScarlet as a reporter protein (Fig. 5a). It required a more concentrated PURE mix to achieve comparable expression levels to their non-fusion versions (see Supplementary Table 4). We observed activity for the GFP fusions of the three EFs, as well as those of the GluRS, CysRS, and IF3. For RRF-GFP, we detected lower activity than for the other candidates, and for IF1 and RF1 fusions, we were not able to discriminate between nascent and backgound activity (Supplementary Figs. 23 and 24). We proceeded to immobilize the genes with the GFP fusions as DNA brushes, each GFP fusion gene in its own brush. We monitored the TIRF signal at the DNA brush compared to the overall fluorescence signal (epi) and observed a strong localization and accumulation of IF1-GFP, IF3-GFP and RF1-GFP to the DNA brushes, as evident by a strong signal at the three brush clusters which was persistent with time (Fig. 5b, c and Supplementary Fig. 25). The DNA brush localization for RRF-GFP, GluRS-GFP, CysRS-GFP and EFs-GFP was mainly observed at the beginning of the reaction (first 20 minutes), after which the TIRF signal at the brushes reached the signal of the surroundings. No specific localization to DNA brushes could be detected for three control proteins, GFP, MBP-GFP and T4-gp25-GFP, even at initial stages of the reaction. We thus concluded that the IF3-, RRF-, GluRS-, CysRS-, and the EFs-GFP fusions are localized to the brush due to their function in translation. Although we did not detect a boost in activity for IF1- and RF1-GFP fusions in bulk solution, we still observed strong localization to their DNA brushes, which we attribute to their RNA-binding activity that was most likely not hampered by the GFP fusion (Supplementary Fig. 25). This hypothesis requires further validation. Active site mutants of IF1 and RF1 fused to GFP also localized to DNA brushes, suggesting that, indeed, localization of these two candidates is due to unspecific RNA binding28,29. Control experiments with purified GFP allowed us to roughly estimate the protein concentrations in the TIRF images, by converting the intensity of the signal at the brush according to the calibration curve (Supplementary Fig. 26). Within the DNA brush, we estimate the GFP fusions of IF1, IF3, and RRF to reach the micromolar range measured within the first hour, while GFP fusions of RF1, CysRS, GluRS, EF-Tu, EF-Ts and EF-G, levels are 10-to-100 fold lower. The localization of the translation protein factors and not those of the control proteins provided direct evidence that protein synthesis from a DNA brush enhances self-regeneration by creating high local concentrations.

Simultaneous self-regeneration of all thirty translation proteins
We attempted the biogenesis of all thirty translation proteins simultaneously in a single reaction by immobilizing mixed DNA brushes on a glass surface (Fig. 6a, b) with relative gene fractions based on the individual self-regeneration (Fig. 1h) and previous DNA brush experiments (Figs. 3 and 4). On the same surface different DNA brushes had an increasing ratio of translation genes, ranging from zero to 90%, keeping constant the GFP-HA fraction at 2% and the overall DNA density by supplementing brushes with DNA coding for an unrelated gene. Our initial attempt to use an initial PURE reaction composed of all the threshold concentrations failed to support GFP-HA synthesis and capture on the surrounding surface (Supplementary Fig. 27), suggesting that these conditions were too stringent. Only with the addition of 25 nM of each IFs and RFs to the initial PURE reaction, with all other Pi at their threshold concentration, we could observe accumulation of GFP-HA on the surface surrounding brushes coding for the translation proteins but not next to brushes with unrelated genes (Fig. 6c–e; Supplementary Fig. 28; Supplementary Tables 2 and 3 and Supplementary Movie 4). To improve the signal-to-background ratio for the measurement of the maximal GFP accumulation rate, we doubled the initial protein concentrations from a 100-fold dilution of the full PURE levels to a 50-fold dilution, with the exception of EF-Tu, which remained at a two-fold dilution from the full level in both versions. The increase in initial protein concentrations accelerated the initial protein synthesis rate (Supplementary Fig. 28). The GFP accumulation rate for the 50-fold diluted PURE peaked at about 3 h compared to a full PURE reaction, which peaked already at 50 min (Supplementary Figs. 29 and 30).

Methods
PURE system preparation
Protein purification. The PURE system was prepared according to Shimizu et al.55: E. coli BL21(DE3) pLyS strain was used for protein expression. All plasmids encoding PURE proteins were obtained from Y. Shimizu. Plasmids were transformed and amplified in E. coli DH5α, except for plasmids coding for AlaRS, AsnRS, ThrRS, IF1-3, EF-G, RRF, CK, T7RNAP, and PheRS, which were transformed to E. coli JM109 due to their expression vector (pQE30). All plasmids were subsequently transformed in E. coli BL21(DE3) pLyS for protein expression. All cultures were grown at 37°C, 250 rpm. Overnight cultures were grown in 5 mL of LB medium (LBX0102, Formedium, UK) with 100 μg/mL of ampicillin or 50 μg/mL of kanamycin. Each strain was inoculated in a flask with 1 L of LB and grown to OD600 = 0.6 before induction with 0.1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG from Applichem, Germany) for 3 h, and then harvested after centrifugation and stored at −80°C.

Protein purification buffer compositions are as follows. Buffer A: 50 mM HEPES-KOH (8042391, Bio-Lab, IL) pH 7.6, 1 M ammonium chloride (3384-12, Avantor, USA), 10 mM MgCl2 and 7 mM 2-mercaptoethanol (b-ME, AlfaAesar, ThermoFisher Scientific, USA, only added immediately before use). Buffer B: 50 mM HEPES-KOH pH 7.6, 100 mM KCl (7447-40-7, Merck, DE), 10 mM MgCl2, 500 mM imidazole (GB9580, Glentham Life Sciences, UK) and 7 mM b-ME. Buffer: 50 mM HEPES-KOH pH 7.6, 100 mM KCl, 10 mM MgCl2, and 7 mM b-ME. Protein storage buffer: 50 mM HEPES-KOH pH 7.6, 100 mM KCl, 10 mM MgCl2, 30% glycerol (007120223300, Bio-Lab, IL) and 7 mM b-ME.

The cell pellets were re-suspended in 30 mL of buffer A and lysed by sonication for 3 min on ice (Sonics Vibra-Cell VC 750; probe tip diameter: 13 mm; 20s:20s pulse; 70% amplitude). Cell debris was removed by centrifugation (25,000 cf, 30 min, 4°C). The protein was then purified from the supernatant either in a gravity-flow column or with an FPLC (Äkta avant, Cytiva, USA). For the gravity-flow method, the supernatant was mixed with 2–3 mL of equilibrated resin (PureCube, Ni-NTA agarose, Cube Biotech) and incubated for up to 2 h at 4°C. After the incubation, lysate was loaded on a column (Econo-Pac, Bio-Rad, USA). The column was washed with 30 mL of a wash buffer (95% buffer A, 5% buffer B) and eluted with 15 mL of an elution buffer (10% buffer A, 90% buffer B). For the FPLC method, the supernatant was loaded onto a 5 mL HisTrap FF Crude column (Cytiva, USA). The column was then washed with 20 column volume of wash buffer (95% buffer A, 5% buffer B). Proteins were eluted by a linear 20 column volume gradient (5 to 100% buffer B) with a flow rate of 1 ml/min. Following either method, the elution fraction was dialyzed twice against HT buffer followed by once against protein storage buffer. Protein purity was assessed with SDS-PAGE gel (4–20% GeBaGel, Gene Bio-Application, IL) and Coomassie staining (InstantBlue, Abcam, UK). The protein solution was concentrated using a Vivaspin 20 centrifugal concentrator (molecular cut-off depending on the protein, Cytiva, USA). Protein concentrations were estimated by absorbance at 280 nm using calculated protein extinction coefficients. Proteins were then stored at −80°C in aliquots.

Ribosome purification. Ribosomes were purified from the E. coli strain A19 (no antibiotics). The cells (1 l culture) were grown from an overnight culture to the OD of 1.2 at 37°C, centrifuged (8000xg, 30 min at 4°C) and resuspended in 45 ml in ribosome buffer A (70 mM KCl, 10 mM MgCl2, 10 mM Tris-HCl, pH 7.4 + 7 mM b-mercaptoethanol) Afterwards, cells were lysed using a French press (Constant Systems Cell Disruptor, England, 12,000 PSI). Protease Inhibitor Mix (1 mM PMSF, 0.4 mM Benzamidine, 0.06 mM Benzamide) is added to the lysate and we centrifugate again (30,000 × g, 1 h, 4°C). We then continued with following three different published protocols.

(1) Anion Exchange Chromatography, as described in Trauner et al.56 using a quaternary amine monolithic column (CIM multus QA 80 ml, 2 μm, BIA separations) connected to an FPLC (Äkta avant, Cytiva, USA). Briefly, the lysate was filtered and loaded to the column, washed (15 ml/min, 2xCV) with ribosome buffer A and then with additional 25% ribosome buffer B (ribosome buffer A+1 M NH4Cl). Ribosome were eluted by a gradient of buffer B (25–65%, 10 ml/min, 80 min) and fractions were collected and concentrated on a Vivaspin 20 3 kDa MWCO with a subsequent buffer exchange to the ribosome storage buffer (20 mM Hepes pH 7.6, 30 mM KCl, 6 mM MgAce, 7 mM b-mercaptoethanol).

(2) Hydrophobic interaction chromatography followed by ultracentrifugation was performed according to Shimizu et al.55. After growth, cell pellets were dissolved in suspension buffer (10 mM Hepes-KOH, pH 7.6, 50 mM KCl, 10 mM MgAce, 7 mM 2-mercaptoethanol) and lysed, 1.5 M ammonium sulfate is added the lysate, and after centrifugation (20,000 xg, 20 min) the supernatant is filtered (0.45 mm). Then the sample is applied to a 5 mL HiTrap Butyl FF column (Cytiva, USA) connected to an FPLC (Äkta avant, Cytiva, USA), washed, eluted (20 mM Hepes-KOH, pH 7.6, 0.75 M ammonium sulfate, 10 mM MgAce, 7 mM 2-mercaptoethanol) and their buffer was exchanged the to ribosome storage buffer. The ribosomes were layered on a sucrose cushion buffer (10 Tris, pH = 8, Mg(OAc)2 14 mM, KOAc 60 mM, Sucrose 1.1 M, b-ME 6 mM) and pelleted using a Ti-70 HT rotor at 311,000 xg (55,000 rpm), 4°C for 17 h.

(3) For the third purification method, we followed the protocol of Halfon et al.57 we first used an ultracentrifuge with a sucrose cushion buffer like previously mentioned, followed by sucrose gradient ultracentrifugation on a 10–40% sucrose gradient with the same buffer 10 Tris, pH = 8, Mg(OAc)2 14 mM, KOAc 60 mM, b-ME 6 mM for 16 h at 62,000 xg in a swing SW-28 rotor (19,000 rpm). Ribosome fractions were collected according to A260 values and pelleted at 200,000 xg (45,000 rpm) using a Ti-70 rotor for 19 h. After purification, the ribosomes from each method were dissolved in or dialyzed against 1× Ribosome buffer (20 mM Hepes, pH = 7.6, 30 mM KCl, 6 mM Mg(OAc)2, 7 mM 2-mercaptoethanol) and stored in aliquots at −80°C.

Mass spectrometry. We measured the protein compositions of three different batches of commercial ribosome solution from (Gene frontier) and three different samples of self-purified ribosomes (see Ribosome purification) (n = 3, different batches for commercial ribosome, and n = 3 from the same batch for self-purified ribosomes). In between runs, we added blanks to further minimize potential carry-over between samples. The samples were subjected to tryptic digestion using an S-trap The resulting peptides were analyzed using Bruker Aurora column on nanoflow liquid chromatography (nanoElute2) coupled to high resolution, high mass accuracy mass spectrometry (timsTOF Pro), in DIA mode. Raw data was processed with Spectronaut software version 18.1. The data was searched against the E. coli proteome database (“Ecoli_UP_Jan25.fasta”), appended with common lab protein contaminants. The quantitative comparisons were calculated using Perseus v1.6.2.3. The intensity values were log-transformed. Missing intensity values were replaced by a zero value. For convenience reasons, all values (intensities and ratios) were transformed back from their log values (since missing values were replaced by zero values. When transferring from log, you will see “1” values).

In addition to fold change values (using geometric means), we performed a student’s t test for these two groups. The resulting p values can help assess the statistical significance of the observed differences in protein abundance. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE58 partner repository with the dataset identifier PXD071320.

Buffer preparation. PURE buffer was prepared by stepwise addition of the following solutions, in the order they are listed here, starting with the volume of water necessary to reach the desired concentrations. The final concentrations are listed in the PURE assembly below. The concentrations listed here are for a 2× buffer stock: 100 mM HEPES-KOH pH 7.6 200 mM K-glutamate, 20 mM Mg(OAc)2, 4 mM ATP, 4 mM GTP, 2 mM CTP, 2 mM UTP, 108 units of A280/mL tRNA, 40 mM creatine phosphate, 2 mM DTT, 20 μg/mL folinic acid, 4 mM spermidine, 0.6 mM each amino acid (prepared according to ref. 59). The solution was aliquoted and stored at −80°C. The optimal Mg(OAc)2 for GFP expression was determined by doing an initial Mg(OAc)2 scan between 10 and 30 mM and varied slightly between 16 and 22 mM between buffer preparation batches.

PURE assembly. The PURE system was mixed on ice. The protein solution contained (following Shimizu et al.55) except for RF2: 704 nM AlaRS, 30 nM ArgRS, 408 nM AsnRS, 119 nM AspRS, 23 nM CysRS, 59 nM GlnRS, 230 nM GluRS, 85 nM GlyRS, 17 nM HisRS, 381 nM IleRS, 41 nM LeuRS, 109 nM LysRS, 27 nM MetRS, 134 nM PheRS, 154 nM ProRS, 38 nM SerRS, 83 nM ThrRS, 29 nM TrpRS, 12 nM TyrRS, 16 nM ValRS, 1.036 μM IF1, 405 nM IF2, 456 nM IF3, 636 nM EF-G, 2.255 μM EF-Tu, 1.588 μM EF-Ts, 247 nM RF1, 162 nM RF3, 458 nM RRF, 568 nM MTF, 89 nM CK, 64 nM NDK, 50 nM T7RNAP., 132 nM MK, 30 nM PPiase; The buffer and energy solution (see “Buffer Preparation”). The ribosomes: 2 μM of commercial (Solution III of PUREfrex 2.1, Gene Frontier, JP) or purified ribosomes. We added to each reaction 1.25% (v/v) GroE mix Gene Frontier, JP and 2.5% (v/v) DnaK mix Gene Frontier, JP, which results in the following concentrations of chaperones: DnaK = 2.5 μM, DnaJ = 0.5 μM, GrpE = 0.5 μM, GroEL = 250 nM and GroES = 500 nM PURE assembly was mostly done by hand pipetting. Initial concentrations of single PURE proteins were diluted by hand in protein storage buffer and then dispensed with a liquid handling robot (iDOT, Dispendix Germany) into a premixed ΔPi-PURE. For the ribosome purity test (Supplementary Fig. 3), we pre-mixed solutions containing 5 proteins by hand, and then we assembled the PURE with an Echo 525 (Labcyte, USA).

DNA preparation
For expression in PURE, all translation genes were placed under a T7 promoter and a strong ribosome binding site (RBS). All genes were cloned into pET-plasmids using Gibson Assembly (NEBuilder HiFi Assembly Master Mix, E2621, NEB, USA). The translation genes have either UGA or UAA stop codons. The UGA stop codon requires RF2, and although RF2 was not added to the PURE mix, its carry-over contamination from the ribosomes was sufficient (Supplementary Fig. 5). Thus there was no observed difference in biogenesis (Δkmax) for genes harboring UGA vs. UAA stop codons. For primers and sequences see Supplementary Data File 1. Plasmids were amplified in E. coli DH5α and purified using Wizard SV-Gel Miniprep (Promega, USA), and DNA concentrations were determined using a NanoDrop (NanoPhotometer, Implen, USA). Linear double-stranded DNA fragments were amplified from plasmids with polymerase chain reaction (PCR) with KAPA HotStart ready mix (07958935001, Roche, Switzerland), using a reverse primer conjugated to biotin35 and a forward primer that was either unmodified or conjugated to an ATTO 647 fluorophore on its 5′ end (Integrated DNA Technologies, USA), and purified with the Wizard SV Gel and PCR Clean-Up System (Promega, USA).

For surface immobilization, DNA was mixed with streptavidin (S4762, Sigma-Aldrich) at a 1.4:1 streptavidin: DNA ratio in 1× phosphate-buffered saline (PBS, 02-023-5A, Sartorius, Germany) and 7% glycerol (Bio-Lab, Israel), forming a DNA-streptavidin conjugate. The correct amplification of DNA fragments and DNA-streptavidin conjugates was verified with 1% agarose gel electrophoresis. The solution deposited on the DASIY-treated surface to create DNA brushes always has a total gene concentration of 150 nM. According to the experiment, this solution was composed of different gene mixes (Supplementary Table 3).

Bulk solution experiments
Bulk expression experiments in the PURE system were conducted at 37°C in a ClarioStar plate reader (BMG Labtech, Germany). DNA was added to the PURE reaction prepared as described above. Volumes of 10 μL were pipetted in a black optical-bottom 384-well plates (Nalge Nunc International, USA), sealed with a sticky transparent foil (SealPlate, Excel Scientific, USA), and spun down at 1000 xg for 30 s. GFP fluorescence was measured with an excitation filter of 470/15 nm, a dichroic filter of 491 nm, and an emission filter of 515–20 nm. Every experiment contained a well with full PURE with the same GFP-DNA concentration for normalization purposes between experiments. For repeats of the same experiment, the critical components of the PURE reaction, such as the protein threshold concentration or the gene DNA, were always added separately to each reaction.

Gel electrophoresis analysis of fluorescently labeled nascent proteins
A more concentrated PURE protein and energy/buffer solution was mixed following the composition provided by PURE version 2 by Kazuta et al.11. Commercial ribosomes (Solution III of PUREfrex 2.1, Gene Frontier, JP) were used according to their published manual. For each PURE gene, 1 nM of DNA and 4% (v/v) Green-Lys tRNA (FluoroTect GreenLys, Promega, USA) were added to the assembled PURE and incubated for 4 h at 37°C in a PCR thermocycler (Bio-Rad, USA). The reactions were incubated for 2 min at 60°C and assessed with SDS-PAGE (160 V 1 h) gel (4–20% GeBaGel, Gene Bio-Application, IL) and imaged with a Typhoon FLA 9000 (GE, USA).

Glass surface experiments
Slide preparation. Fused silica slides (24x24x1 mm, UQG Optics, UK) were coated with a biocompatible photosensitive monolayer34 of a polymer formed by a polyethylene glycol backbone with a protected amine and a triethoxysilyl group at either end. The slides were first cleaned in boiling ethanol (830109326, 96%, Gadot Group, IL) for 10 min, followed by base piranha cleaning (1:1:4 H2O2 (000855032300, Bio-Lab, IL): NH3 (105432, Merck, DE): H2O) at 70°C for 10 min. The slides were incubated for 20 min with a 1 mg ml−1 concentration of polymer dissolved in dried toluene (244511, Sigma-Aldrich, USA), then rinsed with toluene (Bio-Lab, Israel) and dried. Surface amines were then deprotected with UV exposure (UV-KUB, FR, 365 nm, 2.5 J/cm2) through a custom photomask with an array of 30 μm hexagons (CAD/Art Services. USA). The slides were immediately incubated with 0.5 mg/ml biotin 3-sulfo-N-hydroxysuccinimide ester (EZ-link NHS biotin, 20217, ThermoFisher Scientific, USA) in 0.2 M borate-buffered solution pH 8.6 (ThermoFisher Scientific) for 30 min, then rinsed with water and dried. The slides were then fixed on custom fused silica prisms (Zell Quarzglas und Technische Keramik, Germany) with Frame-Seal Slide Chambers adhesive (Bio-Rad, USA). Frame-Seal Slide Chambers of size 15 × 15 mm, with a volume of 65 μL, were applied to the polymer-treated side of the fused silica slides to act as reaction chambers for the DNA brushes.

DNA deposition. The biotinylated surfaces of the wells were patterned with nano-liter DNA-SA droplets using the GIX Microplotter II (Sonoplot Inc., USA) and incubated overnight at room temperature and 50–65% humidity to allow DNA brush build-up through biotin-streptavidin binding. We defined 12 positions for DNA brushes on each chip, ensuring at least a 3 mm difference between positions. We spotted three different identical DNA brushes at each position to increase reproducibility (also referred to as a cluster) and increase GFP signal strength. In each experiment, we titrated the concentration of the PURE genes in the DNA brush cluster in question (e.g., 0, 20, 50, and 90%). The dilutions of the PURE genes were done with a control gene that codes for a non-interacting protein so that the total DNA concentration remained at 150 nM. On each glass slide, we repeated the titration series of DNA brush clusters at least twice.

Capture antibodies immobilization. Biotinylated anti-HA antibodies (50 μg/ml, High Affinity, 12158167001, Roche, Sigma-Aldrich) were mixed with streptavidin (S4762, Sigma-Aldrich) at a concentration ratio of 1.5:1 in 1× PBS and incubated for 30 min at 4°C, after which the mix was diluted to 5 μg/ml of antibodies in 1× PBS. 200 μl of this solution was applied to the surface of the chip after the overnight DNA incubation and incubated for 1 h at 4°C. Afterwards, the surface was washed several times with 1× PBS, then with 50 mM HEPES pH 7.4.

Protein expression from DNA brushes. The HEPES buffer was washed and exchanged with a PURE mix in four consecutive washes of 50 μL each. The well was then sealed with a glass coverslip. The slide and attached prism were immediately positioned on the microscope stage and kept at 17°C with a temperature-controlled holder. The temperature was increased to 37°C to start gene expression. Different experiments on a chip were repeated at least two times.

TIRF imaging
Prior to imaging, the space between the prism and the slide was filled with index-matching liquid (Cargille, USA). The wells were imaged with a custom-made Total Internal Reflection Fluorescence (TIRF) system. An upright microscope (Olympus BX51WI, JP) was equipped with a motorized stage (Scientifica, UK) and a laser system for TIRF excitation. Two lasers (OBIS 488-150 LS and OBIS 647 LX, Coherent, USA) were coupled into a single-mode optical fiber (Ozoptics, CAN), after which the beam was collimated and directed to the prism with a goniometer (Thorlabs, USA) at an angle of total internal reflection on the surface of the chamber, creating an evanescent wave inside the chamber. Images were acquired using an Andor iXon Ultra camera (Andor Technology, UK) and a 10× Olympus objective. The set-up was controlled through a custom-made LabView code (National Instruments, USA). The DNA signal was measured with the red laser at the beginning of the acquisition at 17°C. The GFP signal time series was measured in the green laser afterwards at 37°C.

Data processing and analysis
Plate reader data. Automated data processing and analysis were implemented with Python v3.7, and data was generally handled with the pandas (v2.2.3) package. For bulk solution experiments, fluorescence intensity time traces were normalized:
normFI(t) = (FIi(t) − minFIi) / (maxFIfull − minFIi)
where FI(t) is the fluorescence intensity time trace for sample i, minFIi is the minimum fluorescence intensity value of sample i (background level), and maxFIfull is the maximal value (only after the signal has saturated with time ~4–8 h) of a full PURE reaction from the same experiment with the same concentration of reporter gene. The FI(t) was smoothed by applying the Savitzky–Golay filter (imported from scipy (v.1.14.1) package with a window length=7 and a polynomial order=0) before the first rate is calculated. The normalized max. rate is calculated as the max. rate of the sample divided by the max. rate of a full PURE sample from the same experiment. The second rate is calculated by applying the Savitzky–Golay filter to the first-rate and then calculating its rate again. The normalized second rate is calculated by dividing the max. of the 2nd rate of sample i, by the max. of the 2nd rate of a full sample.

Δkmax is defined as the difference between the normalized max. rate in the presence of the gene and the normalized max. rate in the absence of the gene.

Kendall’s rank correlation coefficient was calculated with the kendalltau function with variant b in the scipy (v.1.14.1) package. For categorical variables (e.g., AaRS: “I” or “II”), the rank-biserial correlation r was calculated with:
r = 1 − (2U) / (n1 n2)
where U is calculated by the Mann–Whitney U test from the scipy (v.1.14.1) package, and n1 and n2 are the sample sizes of the groups that belong to class I or II.

Schematics and illustrations were made with Inkscape.

TIRF microscopy data. For TIRF microscopy images, we averaged the GFP signal of the whole image for each time frame. The background signal was determined to be the green channel signal from one of the first 5 frames and then subtracted from the GFP signal of all other time frames. For experiments with a very low GFP signal compared to the background, we defined two regions of interest (ROI) with the same area. The first ROI-1 is nine circles positioned in the center of the hexagons that contain the antibody traps for GFP-HA (specific GFP signal), and the second ROI-2 is nine circles positioned in areas between hexagons, where the density of the antibodies is significantly reduced (background). The circle positions are chosen to be close to each other within the image to avoid differences in illumination due to the alignment of the laser. The background-subtracted GFP signal for each time frame is then calculated by the mean intensity of ROI-1 minus ROI-2.

For time series of GFP expression from DNA brushes, t=0 is set to the time point when the temperature on the prism holder reached 37°C, determined using a temperature sensor. Image acquisition started after the focus plane had been adjusted at 37°C. This time difference was measured and considered in the data analysis. The TIRF signal from the labeled DNA in the DNA brushes was measured by defining two ROIs of the same area, the first ROI-1, including the DNA brushes (DNA-specific signal), and the second ROI-2, excluding the DNA brushes (background).

The normalization of the GFP signal across glass slides for the TIRF microscopy data was conducted in the following way. For each gene titration series using DNA brushes (e.g., 0%, 10%, 20%…), we used the GFP FI signal from DNA brush without translation genes (“0%”) to normalize the GFP FI signal from the other DNA brushes within this gene titration series reporting on the fold-change in GFP SI signal compared to the “0%” DNA brush. Then, the average and s.e.m. for the fold changes is calculated from the each genetic compositions of DNA brush.

Statistics and reproducibility
No statistical method was used to predetermine sample size, but it was on par with standard biophysical measurements. In plate reader experiments, each reaction was repeated at least three times, and it was made sure that the reactions for the repeats were not prepared or measured on the same day as the first measurement. For TIRF microscopy data, each silica slide contained at least three DNA brushes per cluster, and each cluster was repeated at least once per silica slide. In addition, each measurement was repeated on at least two different silica slides. Data that was determined to be heavily affected by technical problems was discarded after discussion between authors. As standard procedure in our field of study, the experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript focuses on a version of the 2001 PURE translation system of coauthor Shimizu, claiming in the abstract to have \"relieved the bottleneck for protein synthesis...toward autonomous synthetic systems.\" This would be worthy of Nat Com, if really demonstrated.\n\nThe PURE system enables synthesis of individual proteins from 30 translation factors, the ribosome & tRNAs, with various versions using different gene templates. Bottlenecks in efficiencies in the PURE system have prevented researchers from getting anywhere near synthesising a self-replicating system, despite a quarter century of optimisations. The main bottlenecks are protein yields insufficient for \"PURE to make PURE\" 30 translation factors (e.g. refs 4,9,10,25) & an inability so far for \"ribosomes to make ribosomes\" in a purified system, neither of which are addressed in this manuscript.\n\nThe manuscript's main assay (Fig. 1a) is PURE making a fluorescently-labelled protein that, while sensitive & useful for tweaking the system, cannot address the main bottlenecks in efficiency. Even with such a sensitive & limited output, there are still visible issues with yields (e.g. Fig. S1's very weak bands for MetRS, SerRS, Gly33RS & Phe88RS proteins). Ribosome purity is another challenge (e.g. Fig. S2). The authors deserve credit for hard & careful work. However, in my opinion, the bottleneck is not relieved.", "reviewer_2": "Schwarz-Schilling and colleagues demonstrated synthesis of 30 translation proteins in the minimal PURE system, defining the minimal composition of PURE required for its own regeneration. The findings are novel and important for the field.\n\nIn the first part, biogenesis of 20 aaRSs in minimal PURE is investigated. However, the conclusions about the requirements for each aaRS cannot be made without taking into account the amino acid composition of the reporter protein. A correlation analysis between GFP amino acid composition and requirements for each aaRS in the system should be performed and results should be discussed.\n\nIn context of self-regenerating translation system, a comparison of temporal dynamics between self-regenerating ΔPURE with DNA brushes and full PURE with dummy genes and same content of GFP gene DNA brushes would be of interest. When does the self-regenerating system reach its peak GFP production rate in comparison with full PURE? Does production of translation proteins from DNA instead of having set amount of those proteins in full PURE prolong the active protein production phase of PURE? (This follow-up experiment would make the paper more complete and insightful, while even without it I would recommend publication)\n\nOtherwise, the study design is sound, the conclusions are supported by the data presented, method description is detailed and the writing is clear and cohesive.\n\nMinor comments:\nSupplementary figure 4: panel a) - distinguishing between so many shades of this color palette is challenging\npanel c) – Y axis in two sets of panels is different which makes it difficult to compare\nSupplementary figure 5: exact chaperones used should be specified in caption and in the main text upon first mention\nSupplementary figure 13: panel a) – caption error, should specify ‘Right’ and ‘Middle and left’", "reviewer_3": "The manuscript by Matthaeus Schwarz-Schilling and co-workers with the title “Autonomous biogenesis of all thirty translation proteins” reports an attempt to demonstrate the autonomous synthesis of a set of 30 core bacterial protein required for in vitro synthesis of proteins from a DNA template. The system is based on the well-known purified an reconstituted PURE system. The approach the authors chose use the surface immobilized DNA (so-called DNA Brushes). Translation activity is monitored by trapping the synthesized GFP in the surrounding area of the DNA brushes. The majority of the reported work focuses on the demonstration that the de novo synthesized proteins are capable for catalyzing their own synthesis. To this end the authors group the PURE proteins in three classes and assess them by their ability to catalyze GFP synthesis in the absence of one or more if the respective genes (delptaPi). Authors find that different preoteins have different effects on these systems with EF-Tu being the most critical one. The latter should not be surprising giving its role in aa-tRNA binding and delivery to the translating ribosome. The manuscript closes with a final experiment where all 30 proteins are synthesis end off the respective genes in the DNA brushes.\n\nOverall this is an interesting study towards developing self-generating in vitro transcription translation systems as a stepping stone toward artificially cells. The technology and Idea of the DNA Brushes is (although not novel in this context) is exciting, the actual characterization and subsequent discussion are superficial and the impact on the field is not evident. The particular issue that seeding the reaction is still required and that overall this is done in a trial and error approach is not providing generalizability principles that the community could use to build on and develop similar systems. Furthermore, no attempt was undertaken to identify the mechanistic basis for the observed finding of differential dependence in the seeding concentration. In summary, although the authors clearly demonstrate in a very exciting experimental system that they can achieve simultaneous syntheses of all 30 proteins form the minimal translation system in E. coli, transferability is limited. This becomes very clear in the almost superficial discussion.\n\nMinor concerns\n- The authors stress the minimal set of proteins but also include chaperones that are (not clear) are not part of the 30 yet are required.\n- The dependence of the reaction compositions and kinetics on the ribosome preparation is interesting and needs to be analyzed for this to be a generalizable study.\n- Figure 3: Why are no TIRF experiment are showed/performed, corresponding to the other subgroups\n- The author should talk a bit more about the similar approaches to ribosome synthesis\n- What are the yields of the reaction and how could this enable implementation in self-regenerating systems (e.g. artificial cells)\nP 10 l17 “dilution” do the authors mean “deletion”?\nP11 l 17 “amounts” should be “concentrations”\nP11 L17 “The many barriers” The author should provide more detail here. What could be barriers and how does what they find explain this etc.\nP11 L27 “ the localization with the brushes…” confusing please expand.\nP13 L2 “…, the entire translation protein machinery was dependent on high-density immobilized DNA” where was this dependence (directly) demonstrated?"}, "author_response": "Summary of revisions (ordered according to appearance in the manuscript)\n1) Changes to the text:\ni. The title has changed from “Autonomous biogenesis of all thirty translation proteins” to\n“Autonomous self-biogenesis of all thirty proteins of the E. coli translation machinery” for\nemphasis and accuracy.\nii. An additional co-author, Ilan Cohen, has been added for his contribution to the new\nexperiments.\niii. The abstract and introduction were majorly revised to change the conceptual framing of the\nmanuscript. The reviewers’ response, especially that of reviewer 1, made us realize that the\nfrequently used term ‘bottleneck’ created a confusion. We originally used ‘bottleneck’ to\ndescribe a limiting dilute PURE system that we deliberately created as a conceptual approach\nto tackle self-regeneration. However, Reviewer 1 referred to a general bottleneck of the PURE\nsystem to produce proteins at low yields compared to cell lysates. To avoid further confusion\nall the ‘bottlenecks’ were changed to ‘threshold concentration’ referring to the limiting\nconcentration that must be crossed to kick-start self-regeneration.\niv. For similar reasoning, the word ‘seed’ was replaced by either ‘contamination’, ‘initial’ or\n‘threshold concentration’. We originally used the term ‘seed’ to describe a general concept of\na starting material required for self-regeneration. However, this proved confusing, as some\nproteins did not require an externally added ‘seed’ for biogenesis— due to the presence of\ncontaminating protein already in the reaction. We have now clarified the matter by (i)\nreferring to it as ‘contamination’ rather than ‘seed’, (ii) providing direct Mass Spec evidence\nthat contaminations are indeed present in the ribosome preparations.\nv. To gain clarity, we removed the word ‘recovery’ of function and ‘recovery assay’ and directly\nreferred to self-regeneration or biogenesis throughout the manuscript. We also replaced the\n‘recovery score’ with Δk (the difference between the max. rates +/- self-regeneration, i.e.\nmax\ngenes).\nvi. The majorly revised discussion is more elaborate, stressing the general concepts as well as\npoints brought up by the reviewers, including the threshold-and-localization paradigm, the\nnon-additive nature of combined thresholds, and the need for future cost-benefit analysis\nwhen adding auxiliary factors or chaperones. We also discuss the bottleneck that was brought\nup by reviewer 1 in a more quantitative way and estimate the yield limits (700 nM total\nprotein vs. 9800 nM required for “PURE-to-make-PURE”), arguing why PURE at threshold is\nthe most promising route forward.\nMinor changes to the text:\ni. We now indicate in the caption of revised Supplementary Fig. 1, the use of a more\nconcentrated PURE version in the PAGE experiment than what we refer to as full PURE (v0) and\nlacks the chaperone mix. Previously it was only mentioned in the Methods section.\nii. We added the exact composition of the chaperone mix in the results section for revised Figure\n1 and in the caption of revised Supplementary Fig. 10.\niii. In response to a comment from reviewer #3, we added a points on ribosome biogenesis in the\nintroduction, p.3 lines 5-12 and further discussed on p. 21, lines 24-28.\nvii. We have added literature references to the revised manuscript, increasing the number of\ncitations from 43 to 53.\n2) New data and analysis has been added:\nSummary: New data in Fig. 1 d-e, Fig. 4 e-g, Supplementary Fig. 2, 3, 4, 5, 6, 10a,18, 19, 23, 24, 25, 28,\n29 and Supplementary Table 3 and 4.\ni. Revised Fig. 1c illustrates the challenge of demonstrating functional biogenesis at low yields\nand introduces the concept of a threshold concentration —the lowest starting level of\ntranslation factors at which reporter expression becomes evident as more nascent and\nfunctional translation factors are produced by the reaction itself.\nii. A numerical simulation to demonstrate the concept of threshold. A model of AsnRS self-\nsynthesis now illustrates the sharp transition that occurs once the threshold is crossed and\nhow it enables to determine biogenesis (revised Fig. 1e, revised Supplement Fig. 2). In this\ncontext, we also show that the biogenesis in a full PURE system is masked by the background\n(revised Fig. 1d.)\niii. Direct Mass Spec analysis of protein contaminations in ribosome preparations. Four ribosome\npurification methods were compared by LC–MS; the commercial preparation proved the least\ncontaminated, confirming its use in this study (revised Supplement Fig. S5). We then added a\nnew correlation analysis between the GFP expression rate for each P-ΔPURE version (‘leakage\ni\nrate’) and the detection of P in differently purified ribosome solutions (revised Supplementary\ni\nFig. 5c).\niv. Demonstration of translation protein localization to DNA brushes by GFP-fusion: we created C-\nterminal GFP fusions for several factors and show their transient accumulation to their own\ngene brushes, providing experimental evidence of increased local concentration at DNA\nbrushes and thus supporting the localization mechanism (revised Fig. 4f–g, revised\nSupplementary Fig. 25). Prior to localization experiments, we tested the functionality of the\nnascent fusions (P-GFP) by expressing them in bulk PURE reactions (in more concentrated\ni\nprotein mix, Supplementary Table 3) using a second reporter protein ‘mScarlet’ (revised Fig.\n4e, revised Supplementary Fig. 23-24).\nv. New analysis and comparison of dilute versus full PURE. Supplementary data now track GFP\nproduction over time, showing that a 2 % mix at threshold sustains synthesis for > 4 h, whereas\nfull PURE stalls after 90 min (revised Supplementary Fig. 28,29).\nMinor changes (new data and analysis):\ni. We extended the ‘ribosome purity test’ to a total of 5 different in-house ribosome\npreparations, in addition to the commercial ribosomes and rearranged the sub-panels (revised\nSupplementary Fig. 3). We also added triplicates for the commercial ribosomes (revised\nSupplementary Fig. 4).\nii. We have added a quantification of the GFP yield and the max. synthesis rate for our full PURE\nversion in bulk solution (revised Supplementary Fig. 6).\niii. We have added a chaperone mix titration experiment for the biogenesis of ArgRS that shows\nthe optimal chaperone concentrations (revised Supplementary Fig. 10a).\niv. We added a matrix of the amino acid composition for all the aaRS and reporter proteins as part\nof our response to reviewer #2 about the amino acid composition and requirements for the\nbiogenesis of the aaRS (revised Supplementary Fig. 18).\nv. For the correlation analysis between the Green-Lys PAGE band intensity and Δk ,\nmax\nwe now\nnormalize the band intensity by the number of Lys in each protein, which increases the\ncorrelation, quantified by Kendall’s rank coefficient, from τ=0.13 to τ=0.34 (revised\nSupplementary Fig. 19). The data is now fitted with a 1st degree polynomial. This signifies a\nmoderate positive correlation.\nvi. We added a new Supplementary Table 4 in the Supplementary Information with the DNA\nsequences for all the reporter and dummy proteins used in this study.\n3) Re-structuring and rearrangements:\ni. One more figure was added to a total of five figures and the figures were reorganized.\n◦ Revised: Fig. 1 (elaborated general concept + simulation + single-protein threshold screen\nfor all 30 proteins);\n◦ Fig. 2 (self-regeneration EF-Tu, DNA brush & EF group);\n◦ Fig. 3 (self-regeneration of all 20 aaRS);\n◦ Fig. 4 (self-regeneration of all IF/RF groups + new data on localization of GFP fusions);\n◦ Fig. 5 (self-regeneration of all 30 proteins).\nii. Overview of rearrangements in Supplementary Information:\nSupplementary Fig. (old) → (new):\n2 → 3, 3 → 8, 4 → 7, 5 → 10, 6 → 9, 7 → 11,\n8 → 8, 9 → 19, 10 → 20, 11 → 21, 12 → 12, 13, 13 → 14,\n14 → 17, 15 → 22, 16 → 26, 17 → 27,\nSuppelmentary Video\n1 → 3, 2 → 1, 3 → 2\niii. A new summary of threshold determination for each of the thirty PURE components (revised\nFig. 1f–i). All of the 30 individual translation proteins are presented in one bar plot. Previously\nthe data was distributed over several figures.\niv. In revised Supplementary Fig. 7a, we split the data into different sub-panels, so that there is no\nrequirement to use the color palette. In revised Supplementary Fig. 7b, we adjusted the y-axis\nso that it is uniform for all sub-panels.\nv. The experiment describing the two-step validation of EF-Tu activity was moved from the SI to\nthe main figures (revised Fig. 2a–c) to highlight the importance and challenge of this protein\nfactor.\nvi. We split the data in Supplementary Fig. 12 into revised Supplementary Fig. 12 & 13. Then for\nrevised Supplementary Fig. 12-14, we created new sub-panels (to avoid the color palette) and\nstandardized all y-axis limits (for better comparison).\nvii. The moderate negative correlation between the k and the length of the aaRS was moved\nmax\nfrom the SI to revised Fig. 3b, to highlight this effect, which is relevant for any further efforts to\nachieve autonomous and self-regenerating PURE system.\nviii.The masking of co-biogenesis of 18 aaRS in bulk solution with different PURE reactions at\nthreshold has been now moved from original supplementary Fig. 10b to revised Fig. 3c to\nmotivate the need for confinement and localization for all aaRS simultaneously.\nThe rearrangement and restructuring of the figures are accompanied by corresponding rearrangement\nof the text and supplementary figures.\nPoint-by-point response:\nReviewer #1 (Remarks to the Author):\nThis manuscript focuses on a version of the 2001 PURE translation system of coauthor Shimizu,\nclaiming in the abstract to have \"relieved the bottleneck for protein synthesis...toward autonomous\nsynthetic systems.\" This would be worthy of Nat Com, if really demonstrated.\nThe PURE system enables synthesis of individual proteins from 30 translation factors, the ribosome &\ntRNAs, with various versions using different gene templates. Bottlenecks in efficiencies in the PURE\nsystem have prevented researchers from getting anywhere near synthesising a self-replicating system,\ndespite a quarter century of optimisations.\nThe main bottlenecks are protein yields insufficient for \"PURE to make PURE\" 30 translation factors\n(e.g. refs 4,9,10,25) & an inability so far for \"ribosomes to make ribosomes\" in a purified system,\nneither of which are addressed in this manuscript.\nWe thank the reviewer for these comments, which reflected a misunderstanding stemming from our\npoor choice of wording. We realized that the term ‘bottleneck’ that we used frequently in the original\nmanuscript was the source of the confusion. The term bottleneck is indeed used in the literature to\ndescribe the low protein yields produced by the PURE system as a main hurdle toward achieving self-\nregeneration of all the components of the translation machinery and the ribosome. We proposed a\nsolution to a self-regenerating PURE but not by relieving this capacity ‘bottleneck’.\nRather than relieving this ‘bottleneck’, we proposed an alternative scenario for a self-regenerating\nPURE where the PURE is diluted to such an extent that there is hardly any protein synthesis, just\nbarely to synthesize the PURE components, which than feed into the system and amplify the signal.\nThis allows us to detect the functionality of the nascent proteins against the background activity of the\npurified proteins. Reducing the initial concentration of the system while remaining functional and\nregenerating 30 translation proteins simultaneously is a promising direction towards developing\n“PURE to make PURE”.\nIn the original manuscript we referred to these initial dilute conditions as bottlenecks. To avoid further\nconfusion in the revised manuscript, we refer to these dilute conditions as ‘threshold concentration’\nthat must be crossed to kick-start self-regeneration. All the words ‘bottlenecks’ were replaced by\n‘threshold concentration’, we changed the introduction and discussion to clarify this issue and we\nadded a new scheme (revised Fig. 1c) and a new simulation (revised Fig. 1e) to illustrate this approach.\nThe manuscript's main assay (Fig. 1a) is PURE making a fluorescently-labelled protein that, while\nsensitive & useful for tweaking the system, cannot address the main bottlenecks in efficiency.\nYes, we agree with reviewer that the assay does not address the bottlenecks of proteins yields in the\nPURE. We hope that with a clearer picture of our paradigm in mind, the use of the fluorescent\nreporter protein becomes evident. At a threshold concentration of the initial PURE there’s barely any\nreporter synthesized. For each protein component, we find this threshold concentration by a\nsystematic titration and observe the amplification of the fluorescent reporter signal only when the\ngenes coding for the PURE components were added, hence an indication of self-regeneration. The\nmain text and figures were modified extensively, and new data were added to strengthen our claims\nand communicate the ideas better.\nEven with such a sensitive & limited output, there are still visible issues with yields (e.g. Fig. S1's very\nweak bands for MetRS, SerRS, Gly33RS & Phe88RS proteins).\nWe hope that with the new concept of diluted PURE at threshold concentrations for increased\nsensitivity, the issue of low yields of some of the PURE proteins is not a major issue. The PAGE analysis\nwas just to confirm that the proteins were synthesized to their expected lengths. Here the proteins are\nexpressed in a concentrated PURE mix based on Kazuta et al. We deduced their activity and the fact\nthat they contribute to self-regeneration by the sensitive signal of the reporter protein in a PURE at\nthreshold concentration.\nFurther, it is important to mention that the proteins were visualized by a fluorescent analog of Lysine\n(Green-Lys), meaning the signal is proportional to the frequency of lysine in each protein. Thanks to\nthe comment of the reviewer we realized that it would be better to normalize the band intensity by\nthe number of lysines for the correlation analysis in revised Supplementary Fig. 19. We have made the\nrelevant changes to Supplementary Fig. 19 and its caption.\nRibosome purity is another challenge (e.g. Fig. S2).\nYes, we agree with the reviewer that ribosome purity is a challenge, but only because not all proteins\nare contaminating to the same extent. If ribosomes contained sufficient contaminations of all PURE\nproteins at an appropriate threshold, the PURE reaction could self-regenerate without addition of any\nother components.\nWe originally inferred the presence of contaminations in various ribosome preparations by the GFP\nexpression levels in delta PURE reactions. In the revised manuscript we added direct validation using\nmass spectrometry, identifying nearly all translation proteins in the purified ribosomes and\nsignificantly less in the commercially available ribosomes (revised Supplementary Fig. 5). The results\nalso show that the initiation factors are more abundant than other proteins, which explains the strong\ngene expression even when they are removed (revised Fig. 1 and Supplementary Fig. 4).\nThe authors deserve credit for hard & careful work. However, in my opinion, the bottleneck is not\nrelieved.\nWe thank the reviewer for the acknowledgment of the work that went into this study. We hope to\nhave clarified that we did not seek to relieve the bottleneck of the PURE here, which is indeed a major\nchallenge, but that we present a different strategy by carefully identifying the sensitive points of the\nPURE system and by using physical localization and feedback to demonstrate the biogenesis of all\ntranslation proteins, individually and simultaneously despite the low yields. We have made major\nchanges to the manuscript to clarify the experimental approach and the conclusions with regard to\nthe reviewers’ comments, which can be tracked in the summary of revisions.\nReviewer #2 (Remarks to the Author):\nSchwarz-Schilling and colleagues demonstrated synthesis of 30 translation proteins in the minimal\nPURE system, defining the minimal composition of PURE required for its own regeneration. The\nfindings are novel and important for the field.\nIn the first part, biogenesis of 20 aaRSs in minimal PURE is investigated. However, the conclusions\nabout the requirements for each aaRS cannot be made without taking into account the amino acid\ncomposition of the reporter protein. A correlation analysis between GFP amino acid composition and\nrequirements for each aaRS in the system should be performed and results should be discussed.\nWe agree with the reviewer that the amino-acid composition could bias the threshold concentration\nthat is required for each of the aaRSs. Such an analysis was already included in the original manuscript\n(original Supplementary Fig. 9, now in revised Supplementary Fig. 19); no significant correlation\nbetween Δk (see below) and amino-acid count in GFP was detected (τ ≈ 0.04, with Kendall's rank\nmax\ncorrelation coefficient), and a moderate negative correlation between Δk the amino acid count in\nmax\nthe aaRS sequence (τ ≈ -0.32), but scoring below the correlation with the length of the protein ( τ ≈\n-0.39) (revised Fig. 4). We note that in the revised manuscript we use Δk , the difference in maximal\nmax\nsynthesis rates with vs. without the cognate gene, to replace “recovery score”, which seemed to\ncreate confusion.\nBased on the reviewer’s comment, we extended our analysis on this matter and added a new revised\nSupplementary Fig. 18, a composition matrix that shows the count of each amino acid for the reporter\nproteins and the aaRS. The matrix highlights the weakest points: Tryptophan (Trp) and Cysteine (Cys).\neGFP contains a single Trp and TrpRS only two and indeed, in ΔTrpRS reactions GFP max. rates falls to\nonly 40 % (Fig. 1i), which is the highest value among the aaRSs. Yet the GFP yield is fully restored when\nthe trpS gene is supplied, which is sufficient to demonstrate functional TrpRS biogenesis.\nIn regard to Cys, our GFP has two Cys and the biogenesis assay of CysRS with GFP worked very well\n(revised Fig. 1i). However, we were able to confirm that the complete absence of Cys in a reporter\nprotein does nullify the biogenesis test (new Supplementary Fig. 24). This can be seen in the results\nfrom a new assay that was used to investigate the co-localization of the translation proteins to the\nDNA brushes. We added C-terminal GFP fusions to some of the translation proteins including CysRS\nand verified whether they remained functional despite the fusion by using a second reporter protein\nmScarlet (Cys-free). We co-expressed mScarlet with different concentrations of CysRS–GFP fusion (7\nCys) or a dummy protein-GFP (7 Cys) genes in ΔCysRS PURE. We observed for an increasing signal in\nthe GFP channel with higher CysRS-GFP gene concentration, no GFP signal for the dummy protein-GFP,\nand, importantly, no difference in the mScarlet signal between the samples (revised Supplementary\nFig. 24).\nConcerning the requirements for the aaRS, we added new mass spec data of the purified ribosomes,\nwhich shows direct evidence of aaRS contamination (revised Supplementary Fig. 5). But their\ndetection in LC/MS fails to provide a significant correlation with the GFP ‘leakage rate’ in the ΔPURE\nversions, meaning that their threshold concentration might even be below the detection limit of the\nLC/MS. We mention on p.8 line 18 the fact that only GlyRS has threshold concentration that is well\nabove the contamination levels.\nIn context of self-regenerating translation system, a comparison of temporal dynamics between self-\nregenerating ΔPURE with DNA brushes and full PURE with dummy genes and same content of GFP\ngene DNA brushes would be of interest. When does the self-regenerating system reach its peak GFP\nproduction rate in comparison with full PURE?\nWe thank the reviewer for this insightful comment. We conducted the proposed experiment and\nrevised the manuscript accordingly. The new experiment appears in revised Supplementary Fig. 28.\nIn this experiment we measured the GFP accumulation in a full PURE reaction using TIRF microscopy\nfrom DNA brushes coding for different dummy proteins or for the 30 translation proteins. As dummy\nproteins, we used two candidates of different length and a dummy DNA (no T7 RNAP promoter). All\ncontrol genes produced higher total GFP than the translation-factor genes, confirming that in a\nsaturated reaction (full PURE) reporter dynamics are dominated by resource competition and that\nnascent-factor biogenesis is effectively masked.\nWe then added a new analysis to compare the temporal dynamics between the full PURE and the\nPURE at threshold (revised Supplementary Fig. 29). The full PURE reached its maximum GFP\nproduction rate at ≈ 50 min and stopped accumulating GFP by ≈ 90 min irrespective of being exposed\nto dummy genes or the 30 translation genes. The PURE at threshold with translation genes peaked at\n≈ 180 min and continued net synthesis for ≥ 4 h.\nDoes production of translation proteins from DNA instead of having set amount of those proteins in\nfull PURE prolong the active protein production phase of PURE?\n(This follow-up experiment would make the paper more complete and insightful, while even without\nit I would recommend publication)\nWe thank the reviewer for this suggestion. As mentioned before, we do measure a significant longer\nproduction phase in the PURE at threshold in the presence of the translation genes in DNA brushes\n(>4h) compared to a full PURE with dummy genes (~1.5h). It seems reasonable to assume that nascent\nproteins that join the pool of active proteins at later times, prolong the production phase of the PURE,\ngiven that we find localization of nascent proteins at the DNA brush (revised Fig. 4).\nHowever, with the current experimental set-up we cannot address how much other factors, such as\nreduced energy consumption of the PURE at threshold, due to lower synthesis rate, prolongs the\nlifetime of the PURE by itself. For this we propose a different experimental set-up such as a chemostat\nthat can sustain gene expression in a steady-state for several hours with controlled boundary\nconditions that can dynamically reduce the amount of purified proteins for identical PURE version,\n+/- self-regeneration. Building the continuous-flow hardware and validating real-time analytics is beyond\nthe scope of this study. We hope this explanation satisfies the reviewer.\nOtherwise, the study design is sound, the conclusions are supported by the data presented, method\ndescription is detailed and the writing is clear and cohesive.\nWe thank the reviewer for this positive assessment and hope that the revised manuscript has\naddressed the major comments.\nMinor comments:\nSupplementary figure 4: panel a) - distinguishing between so many shades of this color palette is\nchallenging\npanel c) – Y axis in two sets of panels is different which makes it difficult to compare\nWe thank the reviewer for this observation and have adjusted the figure accordingly.\n For Supplementary Fig.4a (revised Supplementary Fig. 7a), we have split the data into different\nsub-panels, so that there is no requirement to use the color palette.\n For Supplementary Fig.4c (revised Supplementary Fig. 7b), we have adjusted the y-axis so that\nis uniform for all sub-panels.\n We split the data in Supplementary Fig. 12 into revised Supplementary Fig. 12 & 13. Then for\nrevised Supplementary Fig. 12-14, we created new sub-panels (to avoid the color palette) and\nadjusted all the y-axis for all sub-panels.\nSupplementary figure 5: exact chaperones used should be specified in caption and in the main text\nupon first mention\nWe thank the reviewer for this comment. We added the exact composition of the chaperone mix in\nthe results section of the main text for revised Figure 1, in the caption (revised Supplementary Fig. 10)\nand in the methods section. We have also added a new sub-panel in revised Supplementary Fig. 10a,\nwhich shows how the concentration of the chaperone mix was determined with ArgRS biogenesis.\nSupplementary figure 13: panel a) – caption error, should specify ‘Right’ and ‘Middle and left’\nWe thank the reviewer for catching this error. The data is now in revised Supplementary Fig. 14 and is\nsplit across several sub-panels (see answer above), so that the caption does not include the error\nanymore.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Matthaeus Schwarz-Schilling and co-workers with the title “Autonomous biogenesis\nof all thirty translation proteins” reports an attempt to demonstrate the autonomous synthesis of a\nset of 30 core bacterial protein required for in vitro synthesis of proteins from a DNA template. The\nsystem is based on the well-known purified an reconstituted PURE system. The approach the authors\nchose use the surface immobilized DNA (so-called DNA Brushes). Translation activity is monitored by\ntrapping the synthesized GFP in the surrounding area of the DNA brushes. The majority of the\nreported work focuses on the demonstration that the de novo synthesized proteins are capable for\ncatalyzing their own synthesis. To this end the authors group the PURE proteins in three classes and\nassess them by their ability to catalyze GFP synthesis in the absence of one or more if the respective\ngenes (delptaPi). Authors find that different preoteins have different effects on these systems with EF-\nTu being the most critical one. The latter should not be surprising giving its role in aa-tRNA binding\nand delivery to the translating ribosome. The manuscript closes with a final experiment where all 30\nproteins are synthesis end off the respective genes in the DNA brushes.\nOverall this is an interesting study towards developing self-generating in vitro transcription translation\nsystems as a sutepping stone toward artificially cells.\nThe technology and Idea of the DNA Brushes is (although not novel in this context) is exciting, the\nactual characterization and subsequent discussion are superficial and the impact on the field is not\nevident.\nWe appreciate this assessment and have substantially deepened both the experimental\ncharacterization and the discussion on general impact and deep insight stemming from our results.\nThe manuscript was revised extensively. The specific changes are mentioned in the summary of\nrevisions.\nThe particular issue that seeding the reaction is still required and that overall this is done in a trial and\nerror approach is not providing generalizability principles that the community could use to build on\nand develop similar systems.\nAs identified by the reviewer, this study is aimed “towards developing self-generating in vitro\ntranscription translation systems as a stepping stone towards artificial cells”. Our revised manuscript\nnow puts forward general principles and testable design rules that can be adapted to other systems.\n We introduce the aim of our study in a different conceptual framing. A fully saturated regime\nmay maximize the yield of every component but would prevent rate acceleration due to the\nnascent components, since the system is already at saturation. Rather, we aimed to create a\ndiluted PURE system to such an extent that there is hardly any protein synthesis, just barely to\nsynthesize the PURE components, which than feed into the system and amplify the reporter\nsignal. For improved clarity we replaced the terms ‘bottleneck’ and ‘seed’ with the more\nprecise term ‘threshold concentration’ as the minimum starting level of a component at which\nreporter expression becomes sensitive to additional copies produced in situ. In traditional\nbiochemistry the threshold concentration is a well-defined concept, for example in the context\nof metabolic control analysis (MCA), it is the point where the flux control coefficient (FCC) is\nmaximal.\n We added a new scheme for clarity (revised Fig. 1c) and a simulation (revised Fig. 1e) that\npredicts the threshold concentration by an ODE model that accounts for dilution of initial\nconditions, resource competition and the amino acid sequence of the protein expressed. It\ndemonstrates the heighten sensitivity of the max. synthesis rate for the case of biogenesis\naround the threshold concentration which we exploit throughout this study.\n With new mass spectrometry data of the commercial and three in-house ribosome batches\n(revised Supp. Fig. 5) we directly identified the ribosome solutions as a source of protein\ncontamination that links it to external threshold concentrations. The compiled results show\nthat for most proteins the threshold concentration is sub-nanomolar, so that residual\ncontaminants in ribosome preparations can push the concentration above the threshold even\nin a ΔPURE reaction.\n Practically, any PURE-like system can be diluted until basal expression falls to about 5–10 % of\nthe saturated rate, placing the reaction close to the threshold. Addition of substrates for\nbiogenesis, i.e. the genes, will then push the system to the sensitive region. Here, the total\ntranslation protein concentration of the PURE at threshold was reduced to 14% of a full PURE,\nwhile still demonstrating biogenesis of all 30 proteins simultaneously. This is an important step\nfor the bottom-up synthetic cell community to build-upon and expand.\nFurthermore, no attempt was undertaken to identify the mechanistic basis for the observed finding of\ndifferential dependence in the seeding concentration.\nWe thank the reviewer for the comment about the mechanistic basis for the findings concerning the\nseed concentrations and have now included our analysis in the manuscript accordingly.\nWhile determining the threshold concentrations for each translation factor, we discovered that trace\namounts of those same factors co-purifying with ribosomes can dominate the read-out of our assay.\nThe mechanism is straightforward: proteins whose thresholds is in the pico- to nanomolar range, e.g.\nmost of the twenty aaRSs, are so sensitive that even minute carry-over is sufficient for them to kick-\nstart biogenesis. Consequently, ribosome preparations, and potentially protein preparations as well,\nmust be essentially contaminant-free if one is to determine the actual threshold transition. GlyRS\nrepresents the only exception within the aaRSs, having a threshold concentration high enough so that\nthe GFP expression levels are strongly reduced in a ΔGlyRS-PURE independently of the ribosome\npurification method.\nEF-Tu represents the opposite extreme. Its threshold lies one to two orders of magnitude above that\nof any other translation protein, mirroring its exceptional abundance in vivo (~ 6 % of total cellular\nprotein in E. coli) and in the translation protein pool, with 21 % of the full PURE mix and~ 74 % in our\nPURE mix at threshold. This high requirement poses a stringent bottleneck for autonomous\nregeneration which was only met by physically clustering its gene and the gene product within DNA-\nbrush reaction centers, as demonstrated by a new TIRF/DNA brush localization assay with C-terminal\nGFP fusion to the translation factors (revised Fig. 4).\nThe persistent “leak” in GFP synthesis seen in the ΔIF1-3 PURE reactions is clarified by the new mass-\nspectrometry data that shows their co-purification with every ribosome preparation we tested. The\nrelease factors also produce a measurable background in ΔRF reactions even when LC–MS did not\ndetect them in the ribosome prep, implying that their threshold lies below the MS detection limit or,\nalternatively, that the RFs are not rate-limiting under our assay conditions.\nIn summary, although the authors clearly demonstrate in a very exciting experimental system that\nthey can achieve simultaneous syntheses of all 30 proteins form the minimal translation system in E.\ncoli, transferability is limited. This becomes very clear in the almost superficial discussion.\nWe thank the referee for the acknowledgment and the feedback. We agree that the broader relevance\nshould have been made more explicitly. New data and a revised discussion section now put our\nfindings into general design principles and facilitate the transfer of the conclusions to different\nsettings.\nFirst, physical gene clustering in DNA brushes mirrors natural strategies. Like bacterial nucleoid-\nproximal translation, brush confinement keeps factors where they are needed. Other artificial-cell\ndesigns that cluster genes on large plasmids or via anchoring to specific locations, e.g. at the\nmembrane, could also serve the same purpose.\nSecond, we present a bottom-up approach of only providing the minimum of each translation protein\nwhile maintaining the ability to sustain their biogenesis. The threshold concentration, k and t\nmax max\nreveal important properties of each translation protein in the PURE system and will help the\ncommunity to build-on for developing a fully self-regenerating in-vitro protein synthesis reaction.\nMinor concerns\n- The authors stress the minimal set of proteins but also include chaperones that are (not clear) are\nnot part of the 30 yet are required.\nWe appreciate the reviewer’s observation and have revised the manuscript to clarify how chaperones\nfit within the framework of the minimal set of 30 translation proteins.\nOur definition of the minimal translation set is restricted to the thirty factors that directly participate\nin the process of peptide-bond formation leading to protein synthesis. Chaperones, kinases for energy\nregeneration, T7 RNA polymerase, and other auxiliary enzymes are therefore outside that core.\nNevertheless, protein folding is an indispensable part of biogenesis, and for ArgRS, we found that\nadding an E. coli chaperone mixture accelerates recovery. The same mixture had no measurable effect\non twelve other aaRSs, and we did not detect a folding deficit for the IF, EF or RF groups. Thus, we\nconsider the inclusion of chaperones as a targeted optimization and not an expansion of the minimal\ntranslation set.\nBecause our study focuses on factors that catalyze translation itself, we did not pursue self-\nregeneration of chaperones, kinases or T7 RNA polymerase. We now note explicitly in the discussion\nthat any fully autonomous cell-free system will eventually have to incorporate these modules and that\na systematic cost–benefit analysis will be required before each additional protein is added.\nThe dependence of the reaction compositions and kinetics on the ribosome preparation is interesting\nand needs to be analyzed for this to be a generalizable study.\nWe thank the reviewer for this comment. We added new experiments for the ‘ribosome purity test’\nfor ribosomes purified by just Hydrophobic Interaction Chromatography (HIC) and just a sucrose-\ncushion ultra-centrifugation step to the four other ribosomes preps and rearranged the sub-panels\n(Supplementary Fig. 3). By directly comparing the GFP expression activity for different ΔPURE versions\none can deduce some insights about methods of ribosome purification that are outside the scope of\nthis manuscript: none of the ribosome preps successfully reduced the GFP activity in ΔIF or ΔRF PURE\nreactions and the aaRS provide a better test for contamination since they vary between the preps.\nFrom this one might be able deduce that the sucrose gradient ultracentrifugation step is the best\nmethods for ΔaaRS-PURE. Furthermore, the commercially available ribosomes reduce the gene\nexpression capacity of the ΔPURE reactions the most, meaning that the ribosome purification requires\nan additional step that is not known to us. We added replicates for the ΔPURE reactions with\ncommercially available ribosomes, as they were used throughout this study (revised Supplementary\nFig. 4)\nThe mass spec data that was added to the revised manuscript revealed significant amounts of\ntranslation proteins in the ribosomes, specifically in the home-made purified ones. In the original\nmanuscript we deduced the presence of these contaminations based on the results of expressing GFP\nin ΔPURE versions but this was not verified directly. Additionally, we found most proteins from E. coli\nin the ribosomes solutions (Supplementary Files). We can conclude that ribosome purification needs\nto be improved. However, for the 30 translation proteins, we did not find a significant correlation\nbetween contamination level and self-regeneration (revised Supplementary Fig. 5). The purification\nmethod of the ribosomes is important for the biogenesis of the aaRS and less so for IFs, EFs and RFs.\n- Figure 3: Why are no TIRF experiment are showed/performed, corresponding to the other subgroups\nWe thank the reviewer for their interest. The original manuscript included results from bulk solution\nreactions showing that by creating combinations of IFs and RFs deletions we could create a threshold\nfor these proteins despite their contaminations in the ribosome preparations. We could detect an\nincrease in the GFP production rate upon addition of the corresponding genes, suggesting that\nnascent IFs and RFS are functional. These results were moved from the original Supplementary Fig. 15\nto the main revised Fig. 4, a-d. There was no necessity to perform the TIRF/DNA brush experiment\nbecause we could demonstrate functionality in bulk solution, which is the more generalized scenario.\nThe DNA brushes are required when the protein expression rate in solution is not sufficient and\nrequires enhancement by confinement.\nIn addition, we included new data demonstrating direct localization of some of the translation\nproteins to their DNA brushes. Among the proteins tested we included the initiation and release\nfactors that were assayed in solution in the revised Fig. 4. We found direct observation with TIRF\nmicroscopy for the accumulation of IF1, IF3, RF1 and to a different extent RRF at the DNA brushes.\nIn addition, we hypothesize that the strong and prolonged localization of IF1, IF3 and RF1 is based on\nnon-specific RNA binding, which is supported by DNA brush localization of active-site mutants of IF1\nand RF1 (IF2-GFP was omitted since the construct did not reach detectable expression levels in PURE).\n- The author should talk a bit more about the similar approaches to ribosome synthesis\nWe thank the reviewer for this comment. We have elaborated on similar approaches to the ribosome.\nIn the introduction we have added:\n“In addition, by encoding 26 different ribosomal proteins and assembly factors and one scaffold\nribosomal RNA in such localized reaction centers, we were able to demonstrate the biogenesis,\nhierarchical order, and assembly timeline of the E. coli small ribosomal subunit19. Here, we utilize this\napproach, immobilizing all the genes coding for the thirty protein factors and enzymes of the\ntranslation machinery as DNA brushes and demonstrating their autonomous co-synthesis and\nfunctional integration back into the machinery that created them. Other scenarios to confine and co-\nlocalize the reaction components, for example by packing multiple genes as continuous synthetic\ngenomes in enclosed compartments, could also be envisioned using the same general principles20–23.”\nAt the end of the discussion we added:\n“Ongoing efforts towards the biogenesis of the ribosome in a minimal system 19,37,41,42, the creation of\nsynthetic tRNA molecules43,44 and the replication of genetic material45–48 in addition to the current\nresults, open avenues towards a self-sustaining minimal protein synthesis system that can self-\nregenerate its components, replacing the purified components with synthetic ones.”\n- What are the yields of the reaction and how could this enable implementation in self-regenerating\nsystems (e.g. artificial cells)\nWe thank the reviewer for this comment. We added a quantification of our PURE in comparison to the\ncommercially available PUREfrex 2.0 in the revised Supplementary Fig. 6. In bulk, the reaction\nproduces ~ 700 nM total protein, with a maximum synthesis rate of roughly 7 nM min⁻¹.\nThe full PURE mix contains ~10.5 µM of the thirty translation proteins. Hence the protein yield of one\nbatch run is still an order of magnitude lower in order to close a full self-replication loop. The PURE at\nthreshold reduces the initial concentration of the proteins to 1360 nM, which is about 13% of the full\nlevels. As demonstrated by the translation protein -GFP fusion experiments, the translation proteins\nseem to be localized to DNA brushes, but we do not actually know the local concentration of the\nmachinery at the brush. Therefore it is possible that at the brush the local concentration of nascent\nmachinery and original machinery is not that different between a PURE at threshold or full levels.\nHowever, the PURE at threshold most likely has higher gains in product per original protein, thus being\nmore efficient in self-regeneration which is important aspect for artificial cells\nP 10 l17 “dilution” do the authors mean “deletion”?\nYes, indeed. Fixed. ->“We hypothesized that we could create threshold conditions by removing\nmultiple factors simultaneously from the initial PURE reaction”\nP11 l 17 “amounts” should be “concentrations”\nAccepted and fixed.\nP11 L17 “The many barriers” The author should provide more detail here. What could be barriers and\nhow does what they find explain this etc.\nWe have removed this sentence from the results section as it lacked precision. Instead, we clearly\ndescribe the experimental set-up, added data for the full PURE and present the results:\n“To improve the signal-to background ratio for the measurement of the maximal GFP accumulation\nrate, we slightly increased the initial concentration from roughly 1% of a full PURE reaction to about\n2%, which accelerated the initial protein synthesis (Supplementary Fig. 27). Only EF-Tu remained at\nabout 40% of full level in both versions. The GFP accumulation rate for the 2% diluted PURE peaked\nafter about 3 hours and remained positive for more than 4 hours, compared to a full PURE reaction\nwhich peaked only after 50 minutes and had stopped net accumulation already after 90 minutes.\n(Supplementary Fig. 28, 29).”\nThe word ‘barrier’ has been replaced with threshold concentration. Later in the discussion section we\ncomment:\n“In defining the threshold concentration for each component, we observed a non-additive effect as\nthe threshold for several proteins simultaneously was higher than the sum of the individual ones, and\nt extended.”\nmax\nP11 L27 “ the localization with the brushes…” confusing please expand.\nThis exact sentence at the end of the results section has been removed as it caused confusion. We\nhope that the new experimental part titled “Direct observation of nascent protein localization to gene\nbrushes” (revised Fig. 4) using GFP fused to the translation proteins and the subsequent discussion\nmake the localization to DNA brushes clear now.\nP13 L2 “…, the entire translation protein machinery was dependent on high-density immobilized DNA”\nwhere was this dependence (directly) demonstrated?\nWe removed this sentence in the discussion as it was not well formulated. In the revised manuscript\nwe have rearranged the results section so that it is clear where the TIRF/DNA brush assay helped us to\ndemonstrating the biogenesis of certain proteins or protein groups (e.g EF-Tu in revised Fig. 2).\nFurther, the sentence, which was at the beginning of the discussion, was replaced by:\n“As a consequence of the initial low rates due to the threshold concentrations, we employed a\nmechanism to catalyze and enhance the efficiency of protein synthesis. Specifically, we utilized gene\nbrushes as reaction centers within a large reservoir of resources, co-localizing the gene expression\nmachinery, enhancing both the efficiency of the reaction and sensitivity of detection. “\nReviewer #1 (Remarks to the Author)\nI stand by my critique of the original submission. I do not think my critique has\nbeen meaningfully addressed by the authors in their revision, nor did I expect\nit to be given the experimental challenges of improving the PURE system.\nWe thank the reviewer for the feedback, which helped us to clarify the\nterminology and concept of the manuscript. We note that this study is not about\n‘improving the PURE system’. Besides the addition of a chaperone mix, we are not\nincreasing, changing or modifying the composition of the PURE system to\nincrease the capacity of the PURE system, which is indeed a major challenge.\nOur manuscript demonstrates the autonomous biogenesis of the 30 translation\nproteins of the original PURE components by creating a highly concentrated\nboundary-free compartment at a dense DNA brush coding for all the genes.\nReviewer #4 (Remarks to the Author)\nSchwarz-Schilling and co-workers demonstrated simultaneous synthesis of 30\ncore translation proteins/factors in the in vitro gene expression (PURE) system,\nmaking an essential step towards self-regeneration of such cell-free biogenesis\nmachinery.\nAfter the first revision, the overall clarity has much improved, especially the\nconcept of the paper is now apparent and convincing, thereby justifying the use\nof diluted PURE components rather than full capacity as the initial condition in\norder to test the self-regenerating ability of the system. Moreover, the follow-up\nexperiments adequately answer the original reviewer #2's requests and further\ndeepen their findings.\nAt this point, I only have minor comments/suggestions, which I hope will make\nthe findings further insightful and complete as follows. Otherwise, their\nsophisticated experiment design and given (including supplemental) data well\nsupport the conclusion (also, the detailed method section will be appreciated),\nand thus I would recommend for publication.\nWe thank the reviewer for this recommendation.\n– The 'DNA brush' experiment is a unique and excellent approach developed in\nthe authors' group to enable such challenges. However, further detailed\nexplanations would be more beneficial due to the limited access/knowledge to the\nsystem for the general audience.\n1) The appearance of the different fractions of genes is somewhat confusing (Fig.2\ng-l, Fig.3 e-f, Fig.5 b-e, Fig. S21, S26, S27, S28).\nWhat is the reference ('100%') concentration of different fractions (percentages)\nof genes? e.g., '10%' means that 10% of 150 nM (15 nM) of a gene used for DNA\nbrush preparation (as in the method ‘DNA deposition’), or 10% of the DNA\nconcentration shown in Table S1-3? This point should be clarified in the main\ntext/figure legends.\nWe thank the reviewer for this remark. Indeed, we assemble high density DNA\nbrushes from droplets with total DNA bulk concentration of 150 nM. Given\nsimilar DNA length of various genes in solution, the DNA brush composition, or\nfraction, maintains the solution composition. We now added citations to earlier\nwork characterizing brush assembly.\nTo improve clarity, we added pie charts depicting the gene percentages in the\nDNA brush to the relevant Figures (Fig.3 g,j, Fig.4 e, Fig.6 b, Fig. S22, S26, S27, S28)\nand added the corresponding descriptions to the captions and the Methods\nsection.\nFurthermore, we split Table S2 into two tables: new S2 for the initial\nconcentration of each purified protein in the PURE and new Table S3 for the\ndifferent gene mix solutions with their corresponding gene concentrations, for\nthe DNA brush.\n2) (Also regarding the first point) Authors mentioned that they do not know the\nexact concentration of DNA brush on a chip in response to the reviewer #3;\nhowever, is it, in principle, technically impossible to estimate the local DNA\nconcentration? It seems that at least a rough estimation of DNA on the surface is\nindeed possible, as the authors use ATTO 647-conjugated DNA brushes for\nvisualization purposes. Such information would be insightful for deepening the\nmechanical understanding and exploring other applications of the transcription-\ntranslation enhancement through the local enrichment of PURE components\naround DNA brushes.\nWe thank the reviewer for pointing this out. Previous work in the lab reported a\nDNA brush density for a typical gene (2000 bp) of 100-1000 per µm²,\ndepending\non DNA length and solution concentration, as well as ionic strength (Buxboim et\nal., 2007, Buxboim et al., 2008, Bracha et al., 2013, Bracha et al., 2014). Generally,\nshorter DNA at high concentration and high salt increase density. The length of\nthe DNA fragments coding for the PURE protein’s ranges between 900 and 4000\nbp, in agreement with this quantitative density measurement. At the salt\nconcentrations of the PURE, the height of the DNA brush is about 100-120 nm, the\nsame scale of the TIRF excitation decay length (Bracha et al. 2013, Bracha et al.\n2014). Thus, we can estimate the local concentration of genes in the DNA brush to\nbe about 1-10 μM. As a comparison, the concentration of genes in an E. coli cell is\nabout 10 μM (4000 genes, volume of 0.7 μm3), which is of the same order of\nmagnitude. In bulk experiments, the gene concentration is usually around 1 nM,\nwhich is three orders of magnitude lower. We have added this information and\nadditional discussions about local protein concentrations at appropriate positions\nin the manuscript.\n– Authors reported that GlyRS needs roughly 40% of full PURE concentration for\nself-regeneration, which is exceptionally higher compared to other factors. Could\nauthors discuss any known difficulties in cell-free expression of GlyRS (e.g.,\nHagino et al. (in ref. 16) observed low activity of purified GlyRS, which might\nshow a similar trend to this study, or the authors assume that the cell-free\nexpressed GlyRS is non-functional?)? Any possible solution to this problem (did\nthe authors test the supplementation of chaperon mix for this gene?) would also\nbe beneficial.\nWe thank the reviewer for this remark. Hagino et al. (now ref. 15) recently\npublished their study and showed that the difference between the activity of the\nin situ expressed aaRS compared to the purified one is the highest for GlyRS\namong all 20 aaRS. This could indeed be an explanation for the high threshold\nconcentration of purified GlyRS. We have added this point to the discussion.\nThe chaperones were present for the biogenesis of GlyRS.\n– Although the authors neglected to discuss in the text, some biochemical\nparameters other than AA length of aaRSs seem to have a moderate correlation to\nthe self-regeneration property in Fig. S19. In particular, the AA hydrophobicity\nmay exhibit a reasonable trend with kmax (aaRSs for more hydrophilic AA have\nhigher activities), which could be an important factor for self-regeneration\nactivity.\nWe thank the reviewer for this comment. We now mention in the main text the\ncorrelation with hydrophobicity of the substrate amino acids and the band\nintensity of each protein expressed in the PURE system. Understanding the origin\nof correlation between the hydrophobicity of the substrate amino acid and self-\nregeneration is beyond the scope of the present paper.\n– The figure references seem to have mismatches in p.16, l.18 (Fig. '4e' may be\n'4f'), l.20 ('4f' may be '4e'), and l.23 ('4f' may be '4g'), please double-check.\nYes, all correct, thank you. We changed the figure references accordingly.\n– p.16, l.33 'we not did' should be 'we did not'.\nYes, thank you, we changed it to “we did not”\n– Fig. S19: Reference for 'Kendall’s rank correlation coefficient' does not appear\ncorrectly in the legend.\nCorrect, we added the τ symbol.\n– Fig. S20: 'seed' should be 'threshold concentration' in the table.\nYes, thank you, the figure has been changed accordingly.\nReviewer #5 (Remarks to the Author):\nI did not review the original submission of the manuscript, but I have carefully\nread the revised version and the authors’ response letter. Overall, I find this study\ninteresting and commend the authors for undertaking such innovative research.\nMy review focused specifically on the response to Reviewer #3’s comments, as\nrequested by the editorial office.\nI fully agree with the concerns raised by Reviewer #3 — while the study is\nexciting, the current system has several notable limitations, and the potential\napplications remain somewhat unclear. Nevertheless, as a proof-of-concept, I\nbelieve the authors have conducted substantial work and have adequately\naddressed Reviewer #3’s concerns in their revision.\nI support the publication of this manuscript in Nature Communications.\nWe thank the reviewer for the positive comments. We hope to have addressed the\nconcerns raised by Reviewer #3."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I stand by my critique of the original submission. I do not think my critique has been meaningfully addressed by the authors in their revision, nor did I expect it to be given the experimental challenges of improving the PURE system.", "reviewer_4": "Schwarz-Schilling and co-workers demonstrated simultaneous synthesis of 30 core translation proteins/factors in the in vitro gene expression (PURE) system, making an essential step towards self-regeneration of such cell-free biogenesis machinery.\n\nAfter the first revision, the overall clarity has much improved, especially the concept of the paper is now apparent and convincing, thereby justifying the use of diluted PURE components rather than full capacity as the initial condition in order to test the self-regenerating ability of the system. Moreover, the follow-up experiments adequately answer the original reviewer #2's requests and further deepen their findings.\n\nAt this point, I only have minor comments/suggestions, which I hope will make the findings further insightful and complete as follows. Otherwise, their sophisticated experiment design and given (including supplemental) data well support the conclusion (also, the detailed method section will be appreciated), and thus I would recommend for publication.\n\n– The 'DNA brush' experiment is a unique and excellent approach developed in the authors' group to enable such challenges. However, further detailed explanations would be more beneficial due to the limited access/knowledge to the system for the general audience.\n\n1) The appearance of the different fractions of genes is somewhat confusing (Fig.2 g-l, Fig.3 e-f, Fig.5 b-e, Fig. S21, S26, S27, S28).\nWhat is the reference ('100%') concentration of different fractions (percentages) of genes? e.g., '10%' means that 10% of 150 nM (15 nM) of a gene used for DNA brush preparation (as in the method ‘DNA deposition’), or 10% of the DNA concentration shown in Table S1-3? This point should be clarified in the main text/figure legends.\n\n2) (Also regarding the first point) Authors mentioned that they do not know the exact concentration of DNA brush on a chip in response to the reviewer #3; however, is it, in principle, technically impossible to estimate the local DNA concentration? It seems that at least a rough estimation of DNA on the surface is indeed possible, as the authors use ATTO 647-conjugated DNA brushes for visualization purposes. Such information would be insightful for deepening the mechanical understanding and exploring other applications of the transcription-translation enhancement through the local enrichment of PURE components around DNA brushes.\n\n– Authors reported that GlyRS needs roughly 40% of full PURE concentration for self-regeneration, which is exceptionally higher compared to other factors. Could authors discuss any known difficulties in cell-free expression of GlyRS (e.g., Hagino et al. (in ref. 16) observed low activity of purified GlyRS, which might show a similar trend to this study, or the authors assume that the cell-free expressed GlyRS is non-functional?)? Any possible solution to this problem (did the authors test the supplementation of chaperon mix for this gene?) would also be beneficial.\n\n– Although the authors neglected to discuss in the text, some biochemical parameters other than AA length of aaRSs seem to have a moderate correlation to the self-regeneration property in Fig. S19. In particular, the AA hydrophobicity may exhibit a reasonable trend with kmax (aaRSs for more hydrophilic AA have higher activities), which could be an important factor for self-regeneration activity.\n\n– The figure references seem to have mismatches in p.16, l.18 (Fig. '4e' may be '4f'), l.20 ('4f' may be '4e'), and l.23 ('4f' may be '4g'), please double-check.\n\n– p.16, l.33 'we not did' should be 'we did not'.\n\n– Fig. S19: Reference for 'Kendall’s rank correlation coefficient' does not appear correctly in the legend.\n\n– Fig. S20: 'seed' should be 'threshold concentration' in the table.", "reviewer_5": "I did not review the original submission of the manuscript, but I have carefully read the revised version and the authors’ response letter. Overall, I find this study interesting and commend the authors for undertaking such innovative research. My review focused specifically on the response to Reviewer #3’s comments, as requested by the editorial office.\n\nI fully agree with the concerns raised by Reviewer #3 — while the study is exciting, the current system has several notable limitations, and the potential applications remain somewhat unclear. Nevertheless, as a proof-of-concept, I believe the authors have conducted substantial work and have adequately addressed Reviewer #3’s concerns in their revision.\n\nI support the publication of this manuscript in Nature Communications."}, "author_response": "Review file\nSummary of revisions (ordered according to appearance in the manuscript)\n1) Changes to the text:\ni. The title has changed from “Autonomous biogenesis of all thirty translation proteins” to\n“Autonomous self-biogenesis of all thirty proteins of the E. coli translation machinery” for\nemphasis and accuracy.\nii. An additional co-author, Ilan Cohen, has been added for his contribution to the new\nexperiments.\niii. The abstract and introduction were majorly revised to change the conceptual framing of the\nmanuscript. The reviewers’ response, especially that of reviewer 1, made us realize that the\nfrequently used term ‘bottleneck’ created a confusion. We originally used ‘bottleneck’ to\ndescribe a limiting dilute PURE system that we deliberately created as a conceptual approach\nto tackle self-regeneration. However, Reviewer 1 referred to a general bottleneck of the PURE\nsystem to produce proteins at low yields compared to cell lysates. To avoid further confusion\nall the ‘bottlenecks’ were changed to ‘threshold concentration’ referring to the limiting\nconcentration that must be crossed to kick-start self-regeneration.\niv. For similar reasoning, the word ‘seed’ was replaced by either ‘contamination’, ‘initial’ or\n‘threshold concentration’. We originally used the term ‘seed’ to describe a general concept of\na starting material required for self-regeneration. However, this proved confusing, as some\nproteins did not require an externally added ‘seed’ for biogenesis— due to the presence of\ncontaminating protein already in the reaction. We have now clarified the matter by (i)\nreferring to it as ‘contamination’ rather than ‘seed’, (ii) providing direct Mass Spec evidence\nthat contaminations are indeed present in the ribosome preparations.\nv. To gain clarity, we removed the word ‘recovery’ of function and ‘recovery assay’ and directly\nreferred to self-regeneration or biogenesis throughout the manuscript. We also replaced the\n‘recovery score’ with Δk (the difference between the max. rates +/- self-regeneration, i.e.\nmax\ngenes).\nvi. The majorly revised discussion is more elaborate, stressing the general concepts as well as\npoints brought up by the reviewers, including the threshold-and-localization paradigm, the\nnon-additive nature of combined thresholds, and the need for future cost-benefit analysis\nwhen adding auxiliary factors or chaperones. We also discuss the bottleneck that was brought\nup by reviewer 1 in a more quantitative way and estimate the yield limits (700 nM total\nprotein vs. 9800 nM required for “PURE-to-make-PURE”), arguing why PURE at threshold is\nthe most promising route forward.\nMinor changes to the text:\ni. We now indicate in the caption of revised Supplementary Fig. 1, the use of a more\nconcentrated PURE version in the PAGE experiment than what we refer to as full PURE (v0) and\nlacks the chaperone mix. Previously it was only mentioned in the Methods section.\nii. We added the exact composition of the chaperone mix in the results section for revised Figure\n1 and in the caption of revised Supplementary Fig. 10.\niii. In response to a comment from reviewer #3, we added a points on ribosome biogenesis in the\nintroduction, p.3 lines 5-12 and further discussed on p. 21, lines 24-28.\nvii. We have added literature references to the revised manuscript, increasing the number of\ncitations from 43 to 53.\n2) New data and analysis has been added:\nSummary: New data in Fig. 1 d-e, Fig. 4 e-g, Supplementary Fig. 2, 3, 4, 5, 6, 10a,18, 19, 23, 24, 25, 28,\n29 and Supplementary Table 3 and 4.\ni. Revised Fig. 1c illustrates the challenge of demonstrating functional biogenesis at low yields\nand introduces the concept of a threshold concentration —the lowest starting level of\ntranslation factors at which reporter expression becomes evident as more nascent and\nfunctional translation factors are produced by the reaction itself.\nii. A numerical simulation to demonstrate the concept of threshold. A model of AsnRS self-\nsynthesis now illustrates the sharp transition that occurs once the threshold is crossed and\nhow it enables to determine biogenesis (revised Fig. 1e, revised Supplement Fig. 2). In this\ncontext, we also show that the biogenesis in a full PURE system is masked by the background\n(revised Fig. 1d.)\niii. Direct Mass Spec analysis of protein contaminations in ribosome preparations. Four ribosome\npurification methods were compared by LC–MS; the commercial preparation proved the least\ncontaminated, confirming its use in this study (revised Supplement Fig. S5). We then added a\nnew correlation analysis between the GFP expression rate for each P-ΔPURE version (‘leakage\ni\nrate’) and the detection of P in differently purified ribosome solutions (revised Supplementary\ni\nFig. 5c).\niv. Demonstration of translation protein localization to DNA brushes by GFP-fusion: we created C-\nterminal GFP fusions for several factors and show their transient accumulation to their own\ngene brushes, providing experimental evidence of increased local concentration at DNA\nbrushes and thus supporting the localization mechanism (revised Fig. 4f–g, revised\nSupplementary Fig. 25). Prior to localization experiments, we tested the functionality of the\nnascent fusions (P-GFP) by expressing them in bulk PURE reactions (in more concentrated\ni\nprotein mix, Supplementary Table 3) using a second reporter protein ‘mScarlet’ (revised Fig.\n4e, revised Supplementary Fig. 23-24).\nv. New analysis and comparison of dilute versus full PURE. Supplementary data now track GFP\nproduction over time, showing that a 2 % mix at threshold sustains synthesis for > 4 h, whereas\nfull PURE stalls after 90 min (revised Supplementary Fig. 28,29).\nMinor changes (new data and analysis):\ni. We extended the ‘ribosome purity test’ to a total of 5 different in-house ribosome\npreparations, in addition to the commercial ribosomes and rearranged the sub-panels (revised\nSupplementary Fig. 3). We also added triplicates for the commercial ribosomes (revised\nSupplementary Fig. 4).\nii. We have added a quantification of the GFP yield and the max. synthesis rate for our full PURE\nversion in bulk solution (revised Supplementary Fig. 6).\niii. We have added a chaperone mix titration experiment for the biogenesis of ArgRS that shows\nthe optimal chaperone concentrations (revised Supplementary Fig. 10a).\niv. We added a matrix of the amino acid composition for all the aaRS and reporter proteins as part\nof our response to reviewer #2 about the amino acid composition and requirements for the\nbiogenesis of the aaRS (revised Supplementary Fig. 18).\nv. For the correlation analysis between the Green-Lys PAGE band intensity and Δk ,\nmax\nwe now\nnormalize the band intensity by the number of Lys in each protein, which increases the\ncorrelation, quantified by Kendall’s rank coefficient, from τ=0.13 to τ=0.34 (revised\nSupplementary Fig. 19). The data is now fitted with a 1st degree polynomial. This signifies a\nmoderate positive correlation.\nvi. We added a new Supplementary Table 4 in the Supplementary Information with the DNA\nsequences for all the reporter and dummy proteins used in this study.\n3) Re-structuring and rearrangements:\ni. One more figure was added to a total of five figures and the figures were reorganized.\n◦ Revised: Fig. 1 (elaborated general concept + simulation + single-protein threshold screen\nfor all 30 proteins);\n◦ Fig. 2 (self-regeneration EF-Tu, DNA brush & EF group);\n◦ Fig. 3 (self-regeneration of all 20 aaRS);\n◦ Fig. 4 (self-regeneration of all IF/RF groups + new data on localization of GFP fusions);\n◦ Fig. 5 (self-regeneration of all 30 proteins).\nii. Overview of rearrangements in Supplementary Information:\nSupplementary Fig. (old) → (new):\n2 → 3, 3 → 8, 4 → 7, 5 → 10, 6 → 9, 7 → 11,\n8 → 8, 9 → 19, 10 → 20, 11 → 21, 12 → 12, 13, 13 → 14,\n14 → 17, 15 → 22, 16 → 26, 17 → 27,\nSuppelmentary Video\n1 → 3, 2 → 1, 3 → 2\niii. A new summary of threshold determination for each of the thirty PURE components (revised\nFig. 1f–i). All of the 30 individual translation proteins are presented in one bar plot. Previously\nthe data was distributed over several figures.\niv. In revised Supplementary Fig. 7a, we split the data into different sub-panels, so that there is no\nrequirement to use the color palette. In revised Supplementary Fig. 7b, we adjusted the y-axis\nso that it is uniform for all sub-panels.\nv. The experiment describing the two-step validation of EF-Tu activity was moved from the SI to\nthe main figures (revised Fig. 2a–c) to highlight the importance and challenge of this protein\nfactor.\nvi. We split the data in Supplementary Fig. 12 into revised Supplementary Fig. 12 & 13. Then for\nrevised Supplementary Fig. 12-14, we created new sub-panels (to avoid the color palette) and\nstandardized all y-axis limits (for better comparison).\nvii. The moderate negative correlation between the k and the length of the aaRS was moved\nmax\nfrom the SI to revised Fig. 3b, to highlight this effect, which is relevant for any further efforts to\nachieve autonomous and self-regenerating PURE system.\nviii.The masking of co-biogenesis of 18 aaRS in bulk solution with different PURE reactions at\nthreshold has been now moved from original supplementary Fig. 10b to revised Fig. 3c to\nmotivate the need for confinement and localization for all aaRS simultaneously.\nThe rearrangement and restructuring of the figures are accompanied by corresponding rearrangement\nof the text and supplementary figures.\nPoint-by-point response:\nReviewer #1 (Remarks to the Author):\nThis manuscript focuses on a version of the 2001 PURE translation system of coauthor Shimizu,\nclaiming in the abstract to have \"relieved the bottleneck for protein synthesis...toward autonomous\nsynthetic systems.\" This would be worthy of Nat Com, if really demonstrated.\nThe PURE system enables synthesis of individual proteins from 30 translation factors, the ribosome &\ntRNAs, with various versions using different gene templates. Bottlenecks in efficiencies in the PURE\nsystem have prevented researchers from getting anywhere near synthesising a self-replicating system,\ndespite a quarter century of optimisations.\nThe main bottlenecks are protein yields insufficient for \"PURE to make PURE\" 30 translation factors\n(e.g. refs 4,9,10,25) & an inability so far for \"ribosomes to make ribosomes\" in a purified system,\nneither of which are addressed in this manuscript.\nWe thank the reviewer for these comments, which reflected a misunderstanding stemming from our\npoor choice of wording. We realized that the term ‘bottleneck’ that we used frequently in the original\nmanuscript was the source of the confusion. The term bottleneck is indeed used in the literature to\ndescribe the low protein yields produced by the PURE system as a main hurdle toward achieving self-\nregeneration of all the components of the translation machinery and the ribosome. We proposed a\nsolution to a self-regenerating PURE but not by relieving this capacity ‘bottleneck’.\nRather than relieving this ‘bottleneck’, we proposed an alternative scenario for a self-regenerating\nPURE where the PURE is diluted to such an extent that there is hardly any protein synthesis, just\nbarely to synthesize the PURE components, which than feed into the system and amplify the signal.\nThis allows us to detect the functionality of the nascent proteins against the background activity of the\npurified proteins. Reducing the initial concentration of the system while remaining functional and\nregenerating 30 translation proteins simultaneously is a promising direction towards developing\n“PURE to make PURE”.\nIn the original manuscript we referred to these initial dilute conditions as bottlenecks. To avoid further\nconfusion in the revised manuscript, we refer to these dilute conditions as ‘threshold concentration’\nthat must be crossed to kick-start self-regeneration. All the words ‘bottlenecks’ were replaced by\n‘threshold concentration’, we changed the introduction and discussion to clarify this issue and we\nadded a new scheme (revised Fig. 1c) and a new simulation (revised Fig. 1e) to illustrate this approach.\nThe manuscript's main assay (Fig. 1a) is PURE making a fluorescently-labelled protein that, while\nsensitive & useful for tweaking the system, cannot address the main bottlenecks in efficiency.\nYes, we agree with reviewer that the assay does not address the bottlenecks of proteins yields in the\nPURE. We hope that with a clearer picture of our paradigm in mind, the use of the fluorescent\nreporter protein becomes evident. At a threshold concentration of the initial PURE there’s barely any\nreporter synthesized. For each protein component, we find this threshold concentration by a\nsystematic titration and observe the amplification of the fluorescent reporter signal only when the\ngenes coding for the PURE components were added, hence an indication of self-regeneration. The\nmain text and figures were modified extensively, and new data were added to strengthen our claims\nand communicate the ideas better.\nEven with such a sensitive & limited output, there are still visible issues with yields (e.g. Fig. S1's very\nweak bands for MetRS, SerRS, Gly33RS & Phe88RS proteins).\nWe hope that with the new concept of diluted PURE at threshold concentrations for increased\nsensitivity, the issue of low yields of some of the PURE proteins is not a major issue. The PAGE analysis\nwas just to confirm that the proteins were synthesized to their expected lengths. Here the proteins are\nexpressed in a concentrated PURE mix based on Kazuta et al. We deduced their activity and the fact\nthat they contribute to self-regeneration by the sensitive signal of the reporter protein in a PURE at\nthreshold concentration.\nFurther, it is important to mention that the proteins were visualized by a fluorescent analog of Lysine\n(Green-Lys), meaning the signal is proportional to the frequency of lysine in each protein. Thanks to\nthe comment of the reviewer we realized that it would be better to normalize the band intensity by\nthe number of lysines for the correlation analysis in revised Supplementary Fig. 19. We have made the\nrelevant changes to Supplementary Fig. 19 and its caption.\nRibosome purity is another challenge (e.g. Fig. S2).\nYes, we agree with the reviewer that ribosome purity is a challenge, but only because not all proteins\nare contaminating to the same extent. If ribosomes contained sufficient contaminations of all PURE\nproteins at an appropriate threshold, the PURE reaction could self-regenerate without addition of any\nother components.\nWe originally inferred the presence of contaminations in various ribosome preparations by the GFP\nexpression levels in delta PURE reactions. In the revised manuscript we added direct validation using\nmass spectrometry, identifying nearly all translation proteins in the purified ribosomes and\nsignificantly less in the commercially available ribosomes (revised Supplementary Fig. 5). The results\nalso show that the initiation factors are more abundant than other proteins, which explains the strong\ngene expression even when they are removed (revised Fig. 1 and Supplementary Fig. 4).\nThe authors deserve credit for hard & careful work. However, in my opinion, the bottleneck is not\nrelieved.\nWe thank the reviewer for the acknowledgment of the work that went into this study. We hope to\nhave clarified that we did not seek to relieve the bottleneck of the PURE here, which is indeed a major\nchallenge, but that we present a different strategy by carefully identifying the sensitive points of the\nPURE system and by using physical localization and feedback to demonstrate the biogenesis of all\ntranslation proteins, individually and simultaneously despite the low yields. We have made major\nchanges to the manuscript to clarify the experimental approach and the conclusions with regard to\nthe reviewers’ comments, which can be tracked in the summary of revisions.\nReviewer #2 (Remarks to the Author):\nSchwarz-Schilling and colleagues demonstrated synthesis of 30 translation proteins in the minimal\nPURE system, defining the minimal composition of PURE required for its own regeneration. The\nfindings are novel and important for the field.\nIn the first part, biogenesis of 20 aaRSs in minimal PURE is investigated. However, the conclusions\nabout the requirements for each aaRS cannot be made without taking into account the amino acid\ncomposition of the reporter protein. A correlation analysis between GFP amino acid composition and\nrequirements for each aaRS in the system should be performed and results should be discussed.\nWe agree with the reviewer that the amino-acid composition could bias the threshold concentration\nthat is required for each of the aaRSs. Such an analysis was already included in the original manuscript\n(original Supplementary Fig. 9, now in revised Supplementary Fig. 19); no significant correlation\nbetween Δk (see below) and amino-acid count in GFP was detected (τ ≈ 0.04, with Kendall's rank\nmax\ncorrelation coefficient), and a moderate negative correlation between Δk the amino acid count in\nmax\nthe aaRS sequence (τ ≈ -0.32), but scoring below the correlation with the length of the protein ( τ ≈\n-0.39) (revised Fig. 4). We note that in the revised manuscript we use Δk , the difference in maximal\nmax\nsynthesis rates with vs. without the cognate gene, to replace “recovery score”, which seemed to\ncreate confusion.\nBased on the reviewer’s comment, we extended our analysis on this matter and added a new revised\nSupplementary Fig. 18, a composition matrix that shows the count of each amino acid for the reporter\nproteins and the aaRS. The matrix highlights the weakest points: Tryptophan (Trp) and Cysteine (Cys).\neGFP contains a single Trp and TrpRS only two and indeed, in ΔTrpRS reactions GFP max. rates falls to\nonly 40 % (Fig. 1i), which is the highest value among the aaRSs. Yet the GFP yield is fully restored when\nthe trpS gene is supplied, which is sufficient to demonstrate functional TrpRS biogenesis.\nIn regard to Cys, our GFP has two Cys and the biogenesis assay of CysRS with GFP worked very well\n(revised Fig. 1i). However, we were able to confirm that the complete absence of Cys in a reporter\nprotein does nullify the biogenesis test (new Supplementary Fig. 24). This can be seen in the results\nfrom a new assay that was used to investigate the co-localization of the translation proteins to the\nDNA brushes. We added C-terminal GFP fusions to some of the translation proteins including CysRS\nand verified whether they remained functional despite the fusion by using a second reporter protein\nmScarlet (Cys-free). We co-expressed mScarlet with different concentrations of CysRS–GFP fusion (7\nCys) or a dummy protein-GFP (7 Cys) genes in ΔCysRS PURE. We observed for an increasing signal in\nthe GFP channel with higher CysRS-GFP gene concentration, no GFP signal for the dummy protein-GFP,\nand, importantly, no difference in the mScarlet signal between the samples (revised Supplementary\nFig. 24).\nConcerning the requirements for the aaRS, we added new mass spec data of the purified ribosomes,\nwhich shows direct evidence of aaRS contamination (revised Supplementary Fig. 5). But their\ndetection in LC/MS fails to provide a significant correlation with the GFP ‘leakage rate’ in the ΔPURE\nversions, meaning that their threshold concentration might even be below the detection limit of the\nLC/MS. We mention on p.8 line 18 the fact that only GlyRS has threshold concentration that is well\nabove the contamination levels.\nIn context of self-regenerating translation system, a comparison of temporal dynamics between self-\nregenerating ΔPURE with DNA brushes and full PURE with dummy genes and same content of GFP\ngene DNA brushes would be of interest. When does the self-regenerating system reach its peak GFP\nproduction rate in comparison with full PURE?\nWe thank the reviewer for this insightful comment. We conducted the proposed experiment and\nrevised the manuscript accordingly. The new experiment appears in revised Supplementary Fig. 28.\nIn this experiment we measured the GFP accumulation in a full PURE reaction using TIRF microscopy\nfrom DNA brushes coding for different dummy proteins or for the 30 translation proteins. As dummy\nproteins, we used two candidates of different length and a dummy DNA (no T7 RNAP promoter). All\ncontrol genes produced higher total GFP than the translation-factor genes, confirming that in a\nsaturated reaction (full PURE) reporter dynamics are dominated by resource competition and that\nnascent-factor biogenesis is effectively masked.\nWe then added a new analysis to compare the temporal dynamics between the full PURE and the\nPURE at threshold (revised Supplementary Fig. 29). The full PURE reached its maximum GFP\nproduction rate at ≈ 50 min and stopped accumulating GFP by ≈ 90 min irrespective of being exposed\nto dummy genes or the 30 translation genes. The PURE at threshold with translation genes peaked at\n≈ 180 min and continued net synthesis for ≥ 4 h.\nDoes production of translation proteins from DNA instead of having set amount of those proteins in\nfull PURE prolong the active protein production phase of PURE?\n(This follow-up experiment would make the paper more complete and insightful, while even without\nit I would recommend publication)\nWe thank the reviewer for this suggestion. As mentioned before, we do measure a significant longer\nproduction phase in the PURE at threshold in the presence of the translation genes in DNA brushes\n(>4h) compared to a full PURE with dummy genes (~1.5h). It seems reasonable to assume that nascent\nproteins that join the pool of active proteins at later times, prolong the production phase of the PURE,\ngiven that we find localization of nascent proteins at the DNA brush (revised Fig. 4).\nHowever, with the current experimental set-up we cannot address how much other factors, such as\nreduced energy consumption of the PURE at threshold, due to lower synthesis rate, prolongs the\nlifetime of the PURE by itself. For this we propose a different experimental set-up such as a chemostat\nthat can sustain gene expression in a steady-state for several hours with controlled boundary\nconditions that can dynamically reduce the amount of purified proteins for identical PURE version,\n+/- self-regeneration. Building the continuous-flow hardware and validating real-time analytics is beyond\nthe scope of this study. We hope this explanation satisfies the reviewer.\nOtherwise, the study design is sound, the conclusions are supported by the data presented, method\ndescription is detailed and the writing is clear and cohesive.\nWe thank the reviewer for this positive assessment and hope that the revised manuscript has\naddressed the major comments.\nMinor comments:\nSupplementary figure 4: panel a) - distinguishing between so many shades of this color palette is\nchallenging\npanel c) – Y axis in two sets of panels is different which makes it difficult to compare\nWe thank the reviewer for this observation and have adjusted the figure accordingly.\n For Supplementary Fig.4a (revised Supplementary Fig. 7a), we have split the data into different\nsub-panels, so that there is no requirement to use the color palette.\n For Supplementary Fig.4c (revised Supplementary Fig. 7b), we have adjusted the y-axis so that\nis uniform for all sub-panels.\n We split the data in Supplementary Fig. 12 into revised Supplementary Fig. 12 & 13. Then for\nrevised Supplementary Fig. 12-14, we created new sub-panels (to avoid the color palette) and\nadjusted all the y-axis for all sub-panels.\nSupplementary figure 5: exact chaperones used should be specified in caption and in the main text\nupon first mention\nWe thank the reviewer for this comment. We added the exact composition of the chaperone mix in\nthe results section of the main text for revised Figure 1, in the caption (revised Supplementary Fig. 10)\nand in the methods section. We have also added a new sub-panel in revised Supplementary Fig. 10a,\nwhich shows how the concentration of the chaperone mix was determined with ArgRS biogenesis.\nSupplementary figure 13: panel a) – caption error, should specify ‘Right’ and ‘Middle and left’\nWe thank the reviewer for catching this error. The data is now in revised Supplementary Fig. 14 and is\nsplit across several sub-panels (see answer above), so that the caption does not include the error\nanymore.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Matthaeus Schwarz-Schilling and co-workers with the title “Autonomous biogenesis\nof all thirty translation proteins” reports an attempt to demonstrate the autonomous synthesis of a\nset of 30 core bacterial protein required for in vitro synthesis of proteins from a DNA template. The\nsystem is based on the well-known purified an reconstituted PURE system. The approach the authors\nchose use the surface immobilized DNA (so-called DNA Brushes). Translation activity is monitored by\ntrapping the synthesized GFP in the surrounding area of the DNA brushes. The majority of the\nreported work focuses on the demonstration that the de novo synthesized proteins are capable for\ncatalyzing their own synthesis. To this end the authors group the PURE proteins in three classes and\nassess them by their ability to catalyze GFP synthesis in the absence of one or more if the respective\ngenes (delptaPi). Authors find that different preoteins have different effects on these systems with EF-\nTu being the most critical one. The latter should not be surprising giving its role in aa-tRNA binding\nand delivery to the translating ribosome. The manuscript closes with a final experiment where all 30\nproteins are synthesis end off the respective genes in the DNA brushes.\nOverall this is an interesting study towards developing self-generating in vitro transcription translation\nsystems as a sutepping stone toward artificially cells.\nThe technology and Idea of the DNA Brushes is (although not novel in this context) is exciting, the\nactual characterization and subsequent discussion are superficial and the impact on the field is not\nevident.\nWe appreciate this assessment and have substantially deepened both the experimental\ncharacterization and the discussion on general impact and deep insight stemming from our results.\nThe manuscript was revised extensively. The specific changes are mentioned in the summary of\nrevisions.\nThe particular issue that seeding the reaction is still required and that overall this is done in a trial and\nerror approach is not providing generalizability principles that the community could use to build on\nand develop similar systems.\nAs identified by the reviewer, this study is aimed “towards developing self-generating in vitro\ntranscription translation systems as a stepping stone towards artificial cells”. Our revised manuscript\nnow puts forward general principles and testable design rules that can be adapted to other systems.\n We introduce the aim of our study in a different conceptual framing. A fully saturated regime\nmay maximize the yield of every component but would prevent rate acceleration due to the\nnascent components, since the system is already at saturation. Rather, we aimed to create a\ndiluted PURE system to such an extent that there is hardly any protein synthesis, just barely to\nsynthesize the PURE components, which than feed into the system and amplify the reporter\nsignal. For improved clarity we replaced the terms ‘bottleneck’ and ‘seed’ with the more\nprecise term ‘threshold concentration’ as the minimum starting level of a component at which\nreporter expression becomes sensitive to additional copies produced in situ. In traditional\nbiochemistry the threshold concentration is a well-defined concept, for example in the context\nof metabolic control analysis (MCA), it is the point where the flux control coefficient (FCC) is\nmaximal.\n We added a new scheme for clarity (revised Fig. 1c) and a simulation (revised Fig. 1e) that\npredicts the threshold concentration by an ODE model that accounts for dilution of initial\nconditions, resource competition and the amino acid sequence of the protein expressed. It\ndemonstrates the heighten sensitivity of the max. synthesis rate for the case of biogenesis\naround the threshold concentration which we exploit throughout this study.\n With new mass spectrometry data of the commercial and three in-house ribosome batches\n(revised Supp. Fig. 5) we directly identified the ribosome solutions as a source of protein\ncontamination that links it to external threshold concentrations. The compiled results show\nthat for most proteins the threshold concentration is sub-nanomolar, so that residual\ncontaminants in ribosome preparations can push the concentration above the threshold even\nin a ΔPURE reaction.\n Practically, any PURE-like system can be diluted until basal expression falls to about 5–10 % of\nthe saturated rate, placing the reaction close to the threshold. Addition of substrates for\nbiogenesis, i.e. the genes, will then push the system to the sensitive region. Here, the total\ntranslation protein concentration of the PURE at threshold was reduced to 14% of a full PURE,\nwhile still demonstrating biogenesis of all 30 proteins simultaneously. This is an important step\nfor the bottom-up synthetic cell community to build-upon and expand.\nFurthermore, no attempt was undertaken to identify the mechanistic basis for the observed finding of\ndifferential dependence in the seeding concentration.\nWe thank the reviewer for the comment about the mechanistic basis for the findings concerning the\nseed concentrations and have now included our analysis in the manuscript accordingly.\nWhile determining the threshold concentrations for each translation factor, we discovered that trace\namounts of those same factors co-purifying with ribosomes can dominate the read-out of our assay.\nThe mechanism is straightforward: proteins whose thresholds is in the pico- to nanomolar range, e.g.\nmost of the twenty aaRSs, are so sensitive that even minute carry-over is sufficient for them to kick-\nstart biogenesis. Consequently, ribosome preparations, and potentially protein preparations as well,\nmust be essentially contaminant-free if one is to determine the actual threshold transition. GlyRS\nrepresents the only exception within the aaRSs, having a threshold concentration high enough so that\nthe GFP expression levels are strongly reduced in a ΔGlyRS-PURE independently of the ribosome\npurification method.\nEF-Tu represents the opposite extreme. Its threshold lies one to two orders of magnitude above that\nof any other translation protein, mirroring its exceptional abundance in vivo (~ 6 % of total cellular\nprotein in E. coli) and in the translation protein pool, with 21 % of the full PURE mix and~ 74 % in our\nPURE mix at threshold. This high requirement poses a stringent bottleneck for autonomous\nregeneration which was only met by physically clustering its gene and the gene product within DNA-\nbrush reaction centers, as demonstrated by a new TIRF/DNA brush localization assay with C-terminal\nGFP fusion to the translation factors (revised Fig. 4).\nThe persistent “leak” in GFP synthesis seen in the ΔIF1-3 PURE reactions is clarified by the new mass-\nspectrometry data that shows their co-purification with every ribosome preparation we tested. The\nrelease factors also produce a measurable background in ΔRF reactions even when LC–MS did not\ndetect them in the ribosome prep, implying that their threshold lies below the MS detection limit or,\nalternatively, that the RFs are not rate-limiting under our assay conditions.\nIn summary, although the authors clearly demonstrate in a very exciting experimental system that\nthey can achieve simultaneous syntheses of all 30 proteins form the minimal translation system in E.\ncoli, transferability is limited. This becomes very clear in the almost superficial discussion.\nWe thank the referee for the acknowledgment and the feedback. We agree that the broader relevance\nshould have been made more explicitly. New data and a revised discussion section now put our\nfindings into general design principles and facilitate the transfer of the conclusions to different\nsettings.\nFirst, physical gene clustering in DNA brushes mirrors natural strategies. Like bacterial nucleoid-\nproximal translation, brush confinement keeps factors where they are needed. Other artificial-cell\ndesigns that cluster genes on large plasmids or via anchoring to specific locations, e.g. at the\nmembrane, could also serve the same purpose.\nSecond, we present a bottom-up approach of only providing the minimum of each translation protein\nwhile maintaining the ability to sustain their biogenesis. The threshold concentration, k and t\nmax max\nreveal important properties of each translation protein in the PURE system and will help the\ncommunity to build-on for developing a fully self-regenerating in-vitro protein synthesis reaction.\nMinor concerns\n- The authors stress the minimal set of proteins but also include chaperones that are (not clear) are\nnot part of the 30 yet are required.\nWe appreciate the reviewer’s observation and have revised the manuscript to clarify how chaperones\nfit within the framework of the minimal set of 30 translation proteins.\nOur definition of the minimal translation set is restricted to the thirty factors that directly participate\nin the process of peptide-bond formation leading to protein synthesis. Chaperones, kinases for energy\nregeneration, T7 RNA polymerase, and other auxiliary enzymes are therefore outside that core.\nNevertheless, protein folding is an indispensable part of biogenesis, and for ArgRS, we found that\nadding an E. coli chaperone mixture accelerates recovery. The same mixture had no measurable effect\non twelve other aaRSs, and we did not detect a folding deficit for the IF, EF or RF groups. Thus, we\nconsider the inclusion of chaperones as a targeted optimization and not an expansion of the minimal\ntranslation set.\nBecause our study focuses on factors that catalyze translation itself, we did not pursue self-\nregeneration of chaperones, kinases or T7 RNA polymerase. We now note explicitly in the discussion\nthat any fully autonomous cell-free system will eventually have to incorporate these modules and that\na systematic cost–benefit analysis will be required before each additional protein is added.\nThe dependence of the reaction compositions and kinetics on the ribosome preparation is interesting\nand needs to be analyzed for this to be a generalizable study.\nWe thank the reviewer for this comment. We added new experiments for the ‘ribosome purity test’\nfor ribosomes purified by just Hydrophobic Interaction Chromatography (HIC) and just a sucrose-\ncushion ultra-centrifugation step to the four other ribosomes preps and rearranged the sub-panels\n(Supplementary Fig. 3). By directly comparing the GFP expression activity for different ΔPURE versions\none can deduce some insights about methods of ribosome purification that are outside the scope of\nthis manuscript: none of the ribosome preps successfully reduced the GFP activity in ΔIF or ΔRF PURE\nreactions and the aaRS provide a better test for contamination since they vary between the preps.\nFrom this one might be able deduce that the sucrose gradient ultracentrifugation step is the best\nmethods for ΔaaRS-PURE. Furthermore, the commercially available ribosomes reduce the gene\nexpression capacity of the ΔPURE reactions the most, meaning that the ribosome purification requires\nan additional step that is not known to us. We added replicates for the ΔPURE reactions with\ncommercially available ribosomes, as they were used throughout this study (revised Supplementary\nFig. 4)\nThe mass spec data that was added to the revised manuscript revealed significant amounts of\ntranslation proteins in the ribosomes, specifically in the home-made purified ones. In the original\nmanuscript we deduced the presence of these contaminations based on the results of expressing GFP\nin ΔPURE versions but this was not verified directly. Additionally, we found most proteins from E. coli\nin the ribosomes solutions (Supplementary Files). We can conclude that ribosome purification needs\nto be improved. However, for the 30 translation proteins, we did not find a significant correlation\nbetween contamination level and self-regeneration (revised Supplementary Fig. 5). The purification\nmethod of the ribosomes is important for the biogenesis of the aaRS and less so for IFs, EFs and RFs.\n- Figure 3: Why are no TIRF experiment are showed/performed, corresponding to the other subgroups\nWe thank the reviewer for their interest. The original manuscript included results from bulk solution\nreactions showing that by creating combinations of IFs and RFs deletions we could create a threshold\nfor these proteins despite their contaminations in the ribosome preparations. We could detect an\nincrease in the GFP production rate upon addition of the corresponding genes, suggesting that\nnascent IFs and RFS are functional. These results were moved from the original Supplementary Fig. 15\nto the main revised Fig. 4, a-d. There was no necessity to perform the TIRF/DNA brush experiment\nbecause we could demonstrate functionality in bulk solution, which is the more generalized scenario.\nThe DNA brushes are required when the protein expression rate in solution is not sufficient and\nrequires enhancement by confinement.\nIn addition, we included new data demonstrating direct localization of some of the translation\nproteins to their DNA brushes. Among the proteins tested we included the initiation and release\nfactors that were assayed in solution in the revised Fig. 4. We found direct observation with TIRF\nmicroscopy for the accumulation of IF1, IF3, RF1 and to a different extent RRF at the DNA brushes.\nIn addition, we hypothesize that the strong and prolonged localization of IF1, IF3 and RF1 is based on\nnon-specific RNA binding, which is supported by DNA brush localization of active-site mutants of IF1\nand RF1 (IF2-GFP was omitted since the construct did not reach detectable expression levels in PURE).\n- The author should talk a bit more about the similar approaches to ribosome synthesis\nWe thank the reviewer for this comment. We have elaborated on similar approaches to the ribosome.\nIn the introduction we have added:\n“In addition, by encoding 26 different ribosomal proteins and assembly factors and one scaffold\nribosomal RNA in such localized reaction centers, we were able to demonstrate the biogenesis,\nhierarchical order, and assembly timeline of the E. coli small ribosomal subunit19. Here, we utilize this\napproach, immobilizing all the genes coding for the thirty protein factors and enzymes of the\ntranslation machinery as DNA brushes and demonstrating their autonomous co-synthesis and\nfunctional integration back into the machinery that created them. Other scenarios to confine and co-\nlocalize the reaction components, for example by packing multiple genes as continuous synthetic\ngenomes in enclosed compartments, could also be envisioned using the same general principles20–23.”\nAt the end of the discussion we added:\n“Ongoing efforts towards the biogenesis of the ribosome in a minimal system 19,37,41,42, the creation of\nsynthetic tRNA molecules43,44 and the replication of genetic material45–48 in addition to the current\nresults, open avenues towards a self-sustaining minimal protein synthesis system that can self-\nregenerate its components, replacing the purified components with synthetic ones.”\n- What are the yields of the reaction and how could this enable implementation in self-regenerating\nsystems (e.g. artificial cells)\nWe thank the reviewer for this comment. We added a quantification of our PURE in comparison to the\ncommercially available PUREfrex 2.0 in the revised Supplementary Fig. 6. In bulk, the reaction\nproduces ~ 700 nM total protein, with a maximum synthesis rate of roughly 7 nM min⁻¹.\nThe full PURE mix contains ~10.5 µM of the thirty translation proteins. Hence the protein yield of one\nbatch run is still an order of magnitude lower in order to close a full self-replication loop. The PURE at\nthreshold reduces the initial concentration of the proteins to 1360 nM, which is about 13% of the full\nlevels. As demonstrated by the translation protein -GFP fusion experiments, the translation proteins\nseem to be localized to DNA brushes, but we do not actually know the local concentration of the\nmachinery at the brush. Therefore it is possible that at the brush the local concentration of nascent\nmachinery and original machinery is not that different between a PURE at threshold or full levels.\nHowever, the PURE at threshold most likely has higher gains in product per original protein, thus being\nmore efficient in self-regeneration which is important aspect for artificial cells\nP 10 l17 “dilution” do the authors mean “deletion”?\nYes, indeed. Fixed. ->“We hypothesized that we could create threshold conditions by removing\nmultiple factors simultaneously from the initial PURE reaction”\nP11 l 17 “amounts” should be “concentrations”\nAccepted and fixed.\nP11 L17 “The many barriers” The author should provide more detail here. What could be barriers and\nhow does what they find explain this etc.\nWe have removed this sentence from the results section as it lacked precision. Instead, we clearly\ndescribe the experimental set-up, added data for the full PURE and present the results:\n“To improve the signal-to background ratio for the measurement of the maximal GFP accumulation\nrate, we slightly increased the initial concentration from roughly 1% of a full PURE reaction to about\n2%, which accelerated the initial protein synthesis (Supplementary Fig. 27). Only EF-Tu remained at\nabout 40% of full level in both versions. The GFP accumulation rate for the 2% diluted PURE peaked\nafter about 3 hours and remained positive for more than 4 hours, compared to a full PURE reaction\nwhich peaked only after 50 minutes and had stopped net accumulation already after 90 minutes.\n(Supplementary Fig. 28, 29).”\nThe word ‘barrier’ has been replaced with threshold concentration. Later in the discussion section we\ncomment:\n“In defining the threshold concentration for each component, we observed a non-additive effect as\nthe threshold for several proteins simultaneously was higher than the sum of the individual ones, and\nt extended.”\nmax\nP11 L27 “ the localization with the brushes…” confusing please expand.\nThis exact sentence at the end of the results section has been removed as it caused confusion. We\nhope that the new experimental part titled “Direct observation of nascent protein localization to gene\nbrushes” (revised Fig. 4) using GFP fused to the translation proteins and the subsequent discussion\nmake the localization to DNA brushes clear now.\nP13 L2 “…, the entire translation protein machinery was dependent on high-density immobilized DNA”\nwhere was this dependence (directly) demonstrated?\nWe removed this sentence in the discussion as it was not well formulated. In the revised manuscript\nwe have rearranged the results section so that it is clear where the TIRF/DNA brush assay helped us to\ndemonstrating the biogenesis of certain proteins or protein groups (e.g EF-Tu in revised Fig. 2).\nFurther, the sentence, which was at the beginning of the discussion, was replaced by:\n“As a consequence of the initial low rates due to the threshold concentrations, we employed a\nmechanism to catalyze and enhance the efficiency of protein synthesis. Specifically, we utilized gene\nbrushes as reaction centers within a large reservoir of resources, co-localizing the gene expression\nmachinery, enhancing both the efficiency of the reaction and sensitivity of detection. “\nReviewer #1 (Remarks to the Author)\nI stand by my critique of the original submission. I do not think my critique has\nbeen meaningfully addressed by the authors in their revision, nor did I expect\nit to be given the experimental challenges of improving the PURE system.\nWe thank the reviewer for the feedback, which helped us to clarify the\nterminology and concept of the manuscript. We note that this study is not about\n‘improving the PURE system’. Besides the addition of a chaperone mix, we are not\nincreasing, changing or modifying the composition of the PURE system to\nincrease the capacity of the PURE system, which is indeed a major challenge.\nOur manuscript demonstrates the autonomous biogenesis of the 30 translation\nproteins of the original PURE components by creating a highly concentrated\nboundary-free compartment at a dense DNA brush coding for all the genes.\nReviewer #4 (Remarks to the Author)\nSchwarz-Schilling and co-workers demonstrated simultaneous synthesis of 30\ncore translation proteins/factors in the in vitro gene expression (PURE) system,\nmaking an essential step towards self-regeneration of such cell-free biogenesis\nmachinery.\nAfter the first revision, the overall clarity has much improved, especially the\nconcept of the paper is now apparent and convincing, thereby justifying the use\nof diluted PURE components rather than full capacity as the initial condition in\norder to test the self-regenerating ability of the system. Moreover, the follow-up\nexperiments adequately answer the original reviewer #2's requests and further\ndeepen their findings.\nAt this point, I only have minor comments/suggestions, which I hope will make\nthe findings further insightful and complete as follows. Otherwise, their\nsophisticated experiment design and given (including supplemental) data well\nsupport the conclusion (also, the detailed method section will be appreciated),\nand thus I would recommend for publication.\nWe thank the reviewer for this recommendation.\n– The 'DNA brush' experiment is a unique and excellent approach developed in\nthe authors' group to enable such challenges. However, further detailed\nexplanations would be more beneficial due to the limited access/knowledge to the\nsystem for the general audience.\n1) The appearance of the different fractions of genes is somewhat confusing (Fig.2\ng-l, Fig.3 e-f, Fig.5 b-e, Fig. S21, S26, S27, S28).\nWhat is the reference ('100%') concentration of different fractions (percentages)\nof genes? e.g., '10%' means that 10% of 150 nM (15 nM) of a gene used for DNA\nbrush preparation (as in the method ‘DNA deposition’), or 10% of the DNA\nconcentration shown in Table S1-3? This point should be clarified in the main\ntext/figure legends.\nWe thank the reviewer for this remark. Indeed, we assemble high density DNA\nbrushes from droplets with total DNA bulk concentration of 150 nM. Given\nsimilar DNA length of various genes in solution, the DNA brush composition, or\nfraction, maintains the solution composition. We now added citations to earlier\nwork characterizing brush assembly.\nTo improve clarity, we added pie charts depicting the gene percentages in the\nDNA brush to the relevant Figures (Fig.3 g,j, Fig.4 e, Fig.6 b, Fig. S22, S26, S27, S28)\nand added the corresponding descriptions to the captions and the Methods\nsection.\nFurthermore, we split Table S2 into two tables: new S2 for the initial\nconcentration of each purified protein in the PURE and new Table S3 for the\ndifferent gene mix solutions with their corresponding gene concentrations, for\nthe DNA brush.\n2) (Also regarding the first point) Authors mentioned that they do not know the\nexact concentration of DNA brush on a chip in response to the reviewer #3;\nhowever, is it, in principle, technically impossible to estimate the local DNA\nconcentration? It seems that at least a rough estimation of DNA on the surface is\nindeed possible, as the authors use ATTO 647-conjugated DNA brushes for\nvisualization purposes. Such information would be insightful for deepening the\nmechanical understanding and exploring other applications of the transcription-\ntranslation enhancement through the local enrichment of PURE components\naround DNA brushes.\nWe thank the reviewer for pointing this out. Previous work in the lab reported a\nDNA brush density for a typical gene (2000 bp) of 100-1000 per µm²,\ndepending\non DNA length and solution concentration, as well as ionic strength (Buxboim et\nal., 2007, Buxboim et al., 2008, Bracha et al., 2013, Bracha et al., 2014). Generally,\nshorter DNA at high concentration and high salt increase density. The length of\nthe DNA fragments coding for the PURE protein’s ranges between 900 and 4000\nbp, in agreement with this quantitative density measurement. At the salt\nconcentrations of the PURE, the height of the DNA brush is about 100-120 nm, the\nsame scale of the TIRF excitation decay length (Bracha et al. 2013, Bracha et al.\n2014). Thus, we can estimate the local concentration of genes in the DNA brush to\nbe about 1-10 μM. As a comparison, the concentration of genes in an E. coli cell is\nabout 10 μM (4000 genes, volume of 0.7 μm3), which is of the same order of\nmagnitude. In bulk experiments, the gene concentration is usually around 1 nM,\nwhich is three orders of magnitude lower. We have added this information and\nadditional discussions about local protein concentrations at appropriate positions\nin the manuscript.\n– Authors reported that GlyRS needs roughly 40% of full PURE concentration for\nself-regeneration, which is exceptionally higher compared to other factors. Could\nauthors discuss any known difficulties in cell-free expression of GlyRS (e.g.,\nHagino et al. (in ref. 16) observed low activity of purified GlyRS, which might\nshow a similar trend to this study, or the authors assume that the cell-free\nexpressed GlyRS is non-functional?)? Any possible solution to this problem (did\nthe authors test the supplementation of chaperon mix for this gene?) would also\nbe beneficial.\nWe thank the reviewer for this remark. Hagino et al. (now ref. 15) recently\npublished their study and showed that the difference between the activity of the\nin situ expressed aaRS compared to the purified one is the highest for GlyRS\namong all 20 aaRS. This could indeed be an explanation for the high threshold\nconcentration of purified GlyRS. We have added this point to the discussion.\nThe chaperones were present for the biogenesis of GlyRS.\n– Although the authors neglected to discuss in the text, some biochemical\nparameters other than AA length of aaRSs seem to have a moderate correlation to\nthe self-regeneration property in Fig. S19. In particular, the AA hydrophobicity\nmay exhibit a reasonable trend with kmax (aaRSs for more hydrophilic AA have\nhigher activities), which could be an important factor for self-regeneration\nactivity.\nWe thank the reviewer for this comment. We now mention in the main text the\ncorrelation with hydrophobicity of the substrate amino acids and the band\nintensity of each protein expressed in the PURE system. Understanding the origin\nof correlation between the hydrophobicity of the substrate amino acid and self-\nregeneration is beyond the scope of the present paper.\n– The figure references seem to have mismatches in p.16, l.18 (Fig. '4e' may be\n'4f'), l.20 ('4f' may be '4e'), and l.23 ('4f' may be '4g'), please double-check.\nYes, all correct, thank you. We changed the figure references accordingly.\n– p.16, l.33 'we not did' should be 'we did not'.\nYes, thank you, we changed it to “we did not”\n– Fig. S19: Reference for 'Kendall’s rank correlation coefficient' does not appear\ncorrectly in the legend.\nCorrect, we added the τ symbol.\n– Fig. S20: 'seed' should be 'threshold concentration' in the table.\nYes, thank you, the figure has been changed accordingly.\nReviewer #5 (Remarks to the Author):\nI did not review the original submission of the manuscript, but I have carefully\nread the revised version and the authors’ response letter. Overall, I find this study\ninteresting and commend the authors for undertaking such innovative research.\nMy review focused specifically on the response to Reviewer #3’s comments, as\nrequested by the editorial office.\nI fully agree with the concerns raised by Reviewer #3 — while the study is\nexciting, the current system has several notable limitations, and the potential\napplications remain somewhat unclear. Nevertheless, as a proof-of-concept, I\nbelieve the authors have conducted substantial work and have adequately\naddressed Reviewer #3’s concerns in their revision.\nI support the publication of this manuscript in Nature Communications.\nWe thank the reviewer for the positive comments. We hope to have addressed the\nconcerns raised by Reviewer #3."}]

Decision letter