A synthetic cell phage cycle

Domain: physics
Published: 2025-12-15
Editorial outcome: minor_revision

Abstract
Viral infection of living cells, exemplified by bacteriophage interaction with bacteria, is fundamental to biology and universal across living systems. Here, we establish an all-cell-free viral cycle where T7 phages infect synthetic cells, equipped with lipopolysaccharides on the outer leaflet of the lipid membrane, while encapsulating a cell-free gene expression system. We track each cycle step to demonstrate T7 phage-specific adsorption onto the liposomes, genome entry, replication, expression, and assembly of new infectious virions within the synthetic cells. We quantify key characteristics of the cycle, including the multiplicity of infection, replication efficiency, liposome size constraints, and phage rebinding dynamics. This work establishes a versatile, fully defined in vitro platform for reconstructing and investigating viral infections from individual molecular components.

Introduction
Building biological systems from their molecular constituents enables isolating, quantifying, and characterizing biochemical mechanisms often entangled and thus difficult to probe in vivo. It is now possible to reconstitute complex biological processes in cell-free systems across multiple scales1–3. Cell-free gene expression (CFE) offers a test bed for validating and enriching our understanding of life’s fundamental principles and constructing synthetic life systems4,5. Among these challenges, establishing a phage cycle is particularly relevant due to the critical role of viral processes in biology. As Arthur Kornberg noted fifty years ago, “Despite the extraordinary complexity of the viral life cycle, its dissection and eventual reconstruction with chemically defined components in a cell-free system from start to finish remains an attractive prospect”6. Strikingly, despite our extensive knowledge of phage evolution7–9, ecology10–12, and molecular mechanisms13–15, a complete cell-free phage infection cycle based on a synthetic cell system has not been achieved, underscoring both conceptual and technical frontiers.

Previous studies have provided isolated elements toward this goal. First, phage φX174 genome ejection was achieved in vitro in the presence of a soluble receptor16. Later, the synthesis of infectious φX174 phages from purified components was demonstrated outside cells17, and its genome was synthetically assembled18. Further, phage T5 genome ejection into liposomes displaying the FhuA receptor19–21 showed that phages can be interfaced with single lipid bilayers via their receptor. Phage T7 infects Escherichia coli and is among the most characterized phages22,23. Every step of its infection cycle has been studied24, making it a relevant phage for assembling a synthetic, cell-free viral cycle. T7 is particularly adequate as a model system due to its obligate lytic cycle, minimal reliance on host factors25, and ease of genome engineering26,27. Previous studies have demonstrated the synthesis of infectious T7 phages in CFE systems28 and genome ejection in the presence of purified LPS (lipopolysaccharide)29. However, in contrast to phage T5, T7 genome translocation into liposomes via its LPS receptor has yet to be achieved. Furthermore, no study has integrated all the steps of a phage infection cycle into a single, self-contained framework.

Here, we set out to build a cell-free phage infection cycle (Fig. 1A) composed of the phage T7 and synthetic cells (SCs) (Fig. 1B). Despite the remarkable simplification compared to the in vivo framework, this synthetic platform reconstitutes all the steps of a phage cycle.

Results
Phage T7 binds specifically to SCs displaying rough LPS
We hypothesized that cell-sized single-bilayer liposomes presenting rough LPS on the outer leaflet and encapsulating a CFE system would enable phage T7 binding, genome entry, genome expression, and subsequent synthesis of phage particles (Fig. 1A). This previously untested hypothesis assumes that rough LPS embedded within a single lipid membrane is a sufficient T7 receptor for genome entry into the SCs. However, creating CFE SCs incorporating rough LPS is challenging. LPS have high molecular weights and poor solubility compatibility with phospholipids, and they must remain stable in the membrane without compromising CFE inside the SCs30,31. Moreover, LPS must be incorporated into the outer leaflet while minimizing their presence in the inner leaflet to prevent unintended phage inactivation inside the SCs. Accordingly, we opted for RdLPS (LPS of the Rd chemotype containing only the inner core; Fig. 1A, a short rough LPS type (Supplementary Fig. 1). To enable interaction with this minimal receptor, we used a cognate T7 phage tail fiber mutant that can bind RdLPS in vitro, unlike wild-type T7 (T7 WT), which binds only to RaLPS (containing both inner and outer core; Supplementary Fig. 1). We assumed that our choice of RdLPS, one of the smallest LPS variants of E. coli, would enable stable integration into SCs, due to its greater hydrophobicity compared to larger LPS targets32,33.

Cell-sized liposomes loaded with a CFE reaction were prepared by emulsion transfer34,35 of a ternary lipid mixture comprised of phosphatidylcholine (POPC), phosphatidylethanolamine polyethyleneglycol (PE-PEG), and RdLPS (Fig. 2A, Supplementary Fig. 2). The presence of PE-PEG was critical for preventing aggregation and facilitating the formation of stable liposomes. We explored the membrane composition while maintaining a total lipid concentration of 100 µM (Fig. 2B, Supplementary Fig. 3). A 55/15/30 mol% PC/PE-PEG/LPS mixture was chosen as it yielded a homogeneous and stable population of RdLPS-liposomes with diameters ranging from 1 µm to 50 µm. Notably, this ratio is consistent with reported LPS molar ratios in the E. coli outer membrane36.

Incorporation of RdLPS into the outer leaflet of the lipid membrane and its expected function were assayed using a chimeric protein (GFP-TF*) consisting of superfolder GFP fused to the N-terminus of the C-terminal ‘tip’ domain of the T7 tail fiber mutant (TF*), which includes the S541R mutation responsible for RdLPS recognition (Supplementary Fig. 4)37. Two liposome populations (PC/PE-PEG and PC/PE-PEG/RdLPS) encapsulating CFE reactions were prepared separately. A red fluorescent dextran marker was added to the CFE reaction encapsulated within the RdLPS liposomes. Upon addition of GFP-TF* to a mixture of both populations, the fluorescence localized exclusively to RdLPS-containing membranes, confirming specific binding and stable LPS incorporation (Fig. 2C, Supplementary Figs. 5-8). We confirmed the asymmetrical localization of the RdLPS on the outer leaflet of the lipid membrane (Supplementary Fig. s9-11, Supplementary Text). The presence of RdLPS in the membrane did not inhibit CFE gene expression activity inside the liposomes (Fig. 2D, Supplementary Figs. 12-13).

To further validate this system, the T7-Split-S* phage was engineered from T7 WT using PHEIGES27 to encode RdLPS-specific tail fibers and to tag capsid proteins fluorescently (Fig. 2E, Supplementary Fig. 14, Supplementary Text). A T7-Split-S* titer of 1011 PFU/mL added to the outer solution of the RdLPS SCs resulted in a clear fluorescent phage localization at the membrane. At an initial MOI (multiplicity of infection) of 1, where binding sites are in excess, over 90% of phages bind within the first 5 min, suggesting efficient initial adsorption (Supplementary Fig. 14)38. These findings confirmed RdLPS-specific phage adsorption onto RdLPS SCs.

T7 genome is internalized, expressed, and replicated into SCs
Bacteriophages eject their genomes into bacterial hosts through diverse mechanisms that have been explored theoretically and experimentally39–41. The genome translocation process, partially driven by internal capsid pressure, has been characterized in vivo for several bacteriophages42,43. For instance, phage T5 can eject its genome into single-bilayer liposomes displaying the FhuA protein receptor via the tip of the long non-contractile tail that crosses the lipid membrane19. In contrast, T7’s short tail prevents T7 from crossing the cell membranes. Inner core proteins are ejected and assemble a trans-membrane channel to translocate the genome into the cell44. Additionally, T7 requires its RNA polymerase to fully translocate its genome45. In a buffer solution, T7 spontaneously releases its genome upon incubation with purified rough LPS due to conformational changes in the binding region29. To track both genome internalization and subsequent gene expression inside the SCs, we engineered two T7 phage variants, either the WT or RdLPS-specific tail fibers (T7-mC-WT, T7-mC-S*), each carrying a mcherry reporter gene downstream of the major capsid gene under the control of a T7 promoter (Fig. 3A). We hypothesized that, as observed in bacterial infections, genome entry into SCs would lead to the expression of the T7 RNA polymerase, thereby initiating a cascade of mcherry gene expression. As designed, the addition of T7-mC-S* phages to the external solution of the RdLPS SCs, encapsulating a DNA-free CFE reaction, resulted in a red fluorescence signal accumulation inside the liposomes (Fig. 3B, Supplementary Figs. 15-16, Supplementary Movie 1), indicating successful genome translocation and the expression of T7 genes. The addition of GFP-TF* to the infected RdLPS SCs revealed that while RdLPS were homogeneously distributed across liposomes, not all liposomes were infected due to SCs leak, disruption or inactive CFE (Fig. 3C). Control experiments using T7-mC-WT, T7-WT, and T7-S* did not yield fluorescence above background levels, confirming that genome internalization was RdLPS-dependent (Fig. 3D, Supplementary Fig. 17).

The efficacy of infection depended on the multiplicity of infection (MOI), defined as the ratio of plaque-forming units (PFU) to SCs (Fig. 3E, Supplementary Figs. 18-19, Supplementary Movie 2). At an MOI of approximately 3, fewer than 2% of SCs exhibited fluorescence. In contrast, nearly 70% of SCs were infected at an MOI of 300 (Fig. 3F). Quantitative analysis showed that, at constant phage concentrations in the outer solution, smaller SCs (<10 µm in diameter) exhibited greater mCherry concentration than larger ones (>15 µm in diameter) (Fig. 3G). We performed an analysis of mCherry concentration in SCs as a function of SCs’ radii across four MOI values (3000, 300, 30, and 3). We observed the same trend, with smaller MOI favoring smaller liposomes while large MOI saturates the liposomes with phage genomes (Supplementary Fig. 20). This suggests that this effect is due to the phage concentration and not to noise associated with the cell-free reaction encapsulation method. This suggests also that the efficacy of genome translocation depends on the surface-to-volume ratio, where smaller SCs, despite fewer genome internalized, maintain a greater genomic concentration relative to their volume. We visualized genome entry into the SCs using SYBR Gold DNA intercalating fluorescent dye that does not inhibit CFE (Fig. 3H, Supplementary Figs. 21-24, Supplementary Movie 3). Upon the addition of T7-mC-S* (MOI ≈ 100) in the outer solution, an increase in fluorescence intensity inside the SCs was observed along with the formation of fluorescent clusters (Fig. 3I, Supplementary Fig. 24), indicating genome entry. Over time, these clusters dissipated as mCherry fluorescence increased, suggesting genome replication and subsequent packaging46,47. The observed cluster formation and disappearance likely reflect concatemer formation and processing during phage packaging. In addition, T7 genome replication was visualized with a fluorescent nucleotide encapsulated within the SCs to further understand the cluster dynamics. Consistent with the DNA intercalating agent results, green fluorescent clusters formed over time, followed by mcherry expression (Fig. 3J, Supplementary Figs. 25-28, Supplementary Movie 4). Cluster formation and subsequent dissipation were quantified, indicating genome replication and phage packaging, leading to the transition from heterogeneous to homogeneous fluorescence in SCs (Fig. 3K, Supplementary Figs. 27-29). We controlled for proper transcription in presence of the fluorescent nucleotide and discarded potential photobleaching (Supplementary Fig. 23).

Phage T7 infection and synthesis can be kinetically tracked in SCs
To estimate the genome entry time into the SCs, we used the engineered phages T7-mC-S* and T7-GFP-S*, in which the egfp gene replaces the mcherry gene at the same position in the genome (Supplementary Data 1). We compared the onset of fluorescence for both phages separately, when the genome was either encapsulated into the SCs or when phages were added in the outer solution. This assay provides two independent measures of the genome entry time, estimated to be 100 min and 105 min, respectively (Fig. 4A, Supplementary Fig. 30). This gives a genome entry time between one and two hours. To determine the genome packaging time and synthesis of infectious phage particles, we monitored the kinetics of PFUs per nanoliters at 30°C in bulk and SCs. We chose a nanoliter unit as it better reflects the small total volume of encapsulated CFE reaction into the SCs. The first synthesized infectious phages were observed after one hour. This experiment was also done at 24°C and 37°C (Supplementary Fig. 31). T7 packaging depends on temperature, the greater the temperature the faster the packaging. After one hour between 102 and 103 PFU/nL were obtained at 30°C and 37°C while no PFU were detected at 24°C. Interestingly, similar titers are obtained between bulk and SCs packaging at 30°C. After 5 h of incubation, we found titers in this range with 10±2×104 PFU/nL, 7±3×104 PFU/nL, 2±1×104 PFU/nL in bulk CFE at 24°C, 30°C and 37°C and 9±3×104 PFU/nL in SCs at 30°C. If all the genomes (0.1 nM) initially added to the CFE reaction become a PFU in absence of DNA replication, we expect 6×104 PFU/nL. We obtain slightly higher titer values than expected suggesting a DNA replication factor of at least 1.5 in SCs at 30°C. It is a lower estimation considering that it is difficult to evaluate genome integrity while only viable phage particles are counted with the agar overlay method. Overall, we concluded that phage packaging time starts within the first hour. After one hour, 1–2% of the final titer is attained at 30°C in bulk and in SCs and reaches >50% of final titer after two hours of incubation, while continuing to increase until five hours of incubation. We hence chose a characteristic packaging time of 1–2 h is our SC model at 30°C.

To further investigate phage assembly within the SCs, we first encapsulated the T7 WT genome at various concentrations into RdLPS-coated SCs and compared, after incubation and lysis by osmotic shock, the phage titers to those obtained from batch 10-µL CFE reactions (Fig. 4C, D, Supplementary Figs. 32-33). Infectious phages were detected in both batch and SCs across all tested genome concentrations. At the lowest DNA concentration (10 pM), phage synthesis was more prevalent inside the SCs than in batch mode, likely due to favorable colocalization of gene expression machinery and phage components under confinement as compared to batch reactions. However, at higher concentrations, phage titers were comparable between the two systems, with slightly lower titers observed in SCs (Fig. 4D). Next, to assess the effect of RdLPS incorporation on phage titer, we encapsulated T7-WT or T7-mC-S* genomes (10 pM) inside SCs with or without RdLPS into the membrane (the RdLPS facing toward the outer solution). After incubation and lysis by osmotic shock, the PFU titers per SC were estimated. While similar PFU per SC were obtained for liposomes lacking RdLPS, a 94±4% reduction of infectious RdLPS-susceptible T7-mC-S* phages was observed in RdLPS-containing SCs (Fig. 4E). These findings confirm that infectious phage particles are assembled inside LPS SCs. However, phage titers are reduced for RdLPS-susceptible phages, likely due to inactivation by irreversible phage binding to RdLPS.

To track phage assembly inside RdLPS SCs, we incubated SCs with T7-Split-S* at an MOI of 100. At this MOI, no detectable fluorescence was observed at the membrane during adsorption. However, once the T7-Split-S* genome entered the SCs, the capsid protein fused to GFP11, and the complementary GFP1-10 were concurrently expressed to form fluorescent capsid proteins as a result of split-GFP complementation. We observed large fluorescent clusters, consistent with capsids self-assembly and binding to the genomic concatemer replicons to package the genomes. Further, this approach enabled visualizing genome concatemer packaging maturation into fluorescent phage capsids. Initially, green fluorescence clusters appeared inside the liposomes suggesting DNA entry and replication (Supplementary Movie 5), as fluorescent capsids localized near DNA concatemers (Fig. 4F, Supplementary Figs. 34-35). Over time, the clusters dispersed into smaller fluorescence foci, suggesting concatemer processing into progeny phage particles.

Phage Release via Osmotic Shock
At the final step of the lytic cycle, phages induce lysis of the host once progeny phages are assembled. In bacterial hosts, lysis is first mediated by a lysis cassette, which is typically expressed late in infection and consists of endolysins (which degrade the cell wall), holins (which form pores in the inner membrane), and spanins (which fuse the inner and outer membranes)48,49. This process results in spheroplast formation that spontaneously lysedueto osmotic shock50. Unlike bacteria, which experience lysis in a hypotonic environment, our SCs are incubated in isotonic conditions, making the phage lysis cassette insufficient, by construction, to induce liposome rupture. Indeed, no significant lysis was observed when phage genomes were expressed inside the RdLPS SCs. To determine the concentration of phages synthesized into the SCs when phages are added to the outer solution, we applied an osmotic shock to an active and a dead SC populations. The dead SC population was created by adding Rnase A into the encapsulated CFE reaction, which ensures that no phage is synthesized. We observed 80 times more phages per liposome in the active compared to dead SCs.

We investigated LPS-carrying SCs as a model for the bacterial outer membrane, and SCs devoid of LPS as a model for the bacterial inner membrane. CFE of the T7 holin into PC/PE-PEG SCs led to significant membrane disruption, as evidenced by leakage of a co-encapsulated dextran dye (Fig. 4H). However, in PC/PE-PEG/RdLPS SCs, no dye leakage was observed, indicating that RdLPS inhibits holin-mediated membrane disruption (Fig. 4H, Supplementary Movie 6). Similar results were obtained with holins from other bacteriophages, supporting that LPS prevents holin activity in this system, which is consistent with its natural function in forming pores in the LPS-less inner membrane of bacteria50. To mimic natural phage release conditions, a simple osmotic shock can induce liposome disruption and phage release (Fig. 4C, D, G), replicating the final step of the lytic cycle.

Methods
Synthetic cell experiments
Lipopolysaccharides from Escherichia coli F583 (Rd mutant) (L6893-5MG, Sigma), 16:0-18:1 PC (POPC) (850457, Avanti Polar Lipids), and 16:0 PEG5000 PE (880200, Avanti Polar Lipids) were used. Additional materials included 4 mL glass vials with Teflon-lined caps (600460, Avanti), Posi-Click tubes from Thomas Scientific (1138W14), liquid paraffin (128-04375, Wako), Optical-Quality Imaging Plates (384-well, black-walled, clear bottom, A58941, Thermo Scientific), dNTP set (100 mM, 10297018, Thermo Fisher), SYBR Gold Nucleic Acid Gel Stain (S11494, Thermo Fisher), Fluorescein-12-dCTP (NU-809-FAMX-S, Jena Bioscience), Tetramethylrhodamine-Dextran (3000 MW, Anionic, Lysine Fixable, Thermo Fisher), and Fluorescein isothiocyanate-dextran (46945-100MG-F, Sigma).

Phage spotting and lysate purification
0.22 μm centrifuge filter tubes were from (8160, Costar), v-bottom 96-well plates (701201, NEST), and square petri dishes (688102, Greiner Bio-One). Difco agar (214010, BD), and LB broth (BP1426-500, Fisher). 100 kDa membrane Amicon Ultra-15 centrifugal filter units (UFC910024, Millipore). Centrifuge tubes, culture tubes, and 25 mL reservoirs were generic.

Phage DNA extraction
DNase I recombinant RNAse-free (M0303S, NEB), RNase A (T3018L, NEB), Proteinase K (P8111S, NEB). SDS (20% wt/vol, AM9820, Sigma), Phenol:Chloroform Alcohol (25:24:1, 15593049, Sigma), 3M sodium acetate solution (pH 5.2, AM9740, Sigma). Absolute ethanol and chloroform were generic, as available.

Assembly and PCR
The T7 genome was obtained from Boca Scientific (310025). KOD1 polymerase was sourced from Diagnocin (TYB-KMM-101), and a PCR clean-up kit from Invitrogen (K310001). PCR tubes (8 strips) were generic, as available. 10× TAE buffer was purchased from Invitrogen (AM9869), and a 1kb+ DNA ladder from Invitrogen (10-787-018) and Greenglo (NC1620342, Thomas Scientific). For assembly, exonuclease III (M0206S, NEB), PEG8000 from Sigma (P2139), and Tris-HCl 1M (15568-025, Invitrogen). All buffers were prepared freshly, pH adjusted, autoclaved and filter sterilized. Large bore low binding tips were used when handling phage genomic DNA and liposomes.

Engineering and phenotyping of phages T7-mC-S* and T7-Split-S* (Supplementary Fig. 37)
Genome assembly was performed following the PHEIGES workflow method1. Preparation of the 2X assembly buffer should be done in advance, aliquoted into 50 μL tubes, and stored at −20°C. 2X Assembly Buffer: 20 mM Tris-HCl (pH 7.9), 100 mM NaCl, 20 mM MgCl2, 10% (w/v) PEG8000, 2 mM Dithiothreitol (DTT) and 1 U/μL Exonuclease III. Genome Assembly: 2X assembly buffer, DNA fragments, and CFE reaction are thawed on ice. Equal volumes of DNA fragments (1–5 nM) are mixed to create an equimolar DNA mix. T7-mC-S* and T7-Split-S* DNA mixes were prepared following assembly maps in Supplementary Data 1 at 1 nM for each fragment. 2.5 μL of DNA mix is mixed to 2.5 μL of 2X assembly buffer in a 1.5 mL tube on ice. The mixture is homogenized, spin down and incubated 1 min at room temperature. The tube is then transferred on a heat block prewarmed at 75°C for 1 min, then at 25°C for 5 min. 7.5 μL of 75% CFE reaction is added to 2.5 μL of the assembly mix. The CFE reaction is gently mix, spined down and incubate at 29°C for at least 4 h. CFE reactions containing the synthesized T7-mC-S* or T7-Split-S* were then spotted on an E. coli rfaC lawn (RdLPS sensitive tail fiber mutants). Isolated plaques are picked and mixed to a liquid culture of E. coli rfaC at OD = 0.05. 200 µL of this culture containing the phage is incubated at 37°C in a shaking plate reader (Agilent Biotek H1m or Neo2) to follow optical density, GFP and mCherry fluorescence over time. The obtained signals from the plate reader (Agilent Biotek H1m or Neo2) were acquired with the Gen5 software. The data presented shows OD, red and green fluorescence from four wells containing a T7-mC-S* isolated plaque, a T7-Split-S* isolated plaque, only the LB medium and the E. coli rfaC culture without phage. The plate reader (Agilent Biotek H1m or Neo2) was incubated at 37°C, with orbital shaking and signals were recorded every 5 minutes for 6 hours.

Phage stock preparation for SCs experiments
Engineered phage lysates are amplified in 2–5 liter of E. coli rfaC as the host until lysis. Phage lysates are treated with 0.5% chloroform to clear remaining bacteria. The lysates are then filter-sterilized through 0.22 μm filters. Lysates are then washed and purified using 100 kDa ultrafiltration columns in phage buffer (10– mM K-glutamate, 1 mM Mg-glutamate, 10 mM Tris-HCl). Phage lysates are washed until no more fluorescence is detected in the pass-through solution (generally 3–5 10-mL wash). Phage lysates are recovered in 0.5 mL–1 mL, phage titers are recorded (typically reaching 1010–1011 PFU/mL) and the phage stock stored at 4°C (phages stocks are used for SCs experiments within 2–3 weeks).

Phage DNA purification and sequencing
Phage DNA Purification T7 phage genomic DNA was extracted using the phenol-chloroform method2. Briefly, 10 mL of pre-treated lysate (1011 PFU/mL) and 10 mL of phenol-chloroform-isoamyl alcohol (PCI) to a separatory funnel. Invert gently, release pressure via the stopcock, and allow layers to separate (20–30 min). Remove the phenol phase (bottom layer) and interphase. Repeat PCI extraction twice more, then perform a final wash with pure chloroform. Recover the aqueous phase, add 1 mL sodium acetate, and 20 mL pre-chilled ethanol. Incubate at −80°C for 1 h, centrifuge at 4000 x g for 20 min, and wash the DNA pellet with 70% ethanol. Resuspend the DNA pellet in 100 μL deionized water (typically 20 nM of T7 phage is obtained). Measure DNA concentration by Nanodrop. The tree phage genomes generated in this study, sequenced using long-read nanopore sequencing, have been deposited in the European Nucleotide Archive (ENA) under the BioProject PRJEB104191. The genome assemblies correspond to the three engineered variants T7-mC-S*, T7-GFP-S*, and T7-Split-S*, and are available under the accession codes OZ370878, OZ370879, and OZ370880, respectively.

Emulsion transfer method for Synthesis of RdLPS SCs (Supplementary Fig. 2)
The lipid composition to incorporate RdLPS in liposomes is composed of POPC (760.076 g/mol), PEG-PE 5000 (5745.03 g/mol) and RdLPS (considered here 2700 g/mol55). The final concentration in the oil should be in the range: POPC: 25 µM–125 µM, PEG-PE: 5 µM–35 µM RdLPS: 20 µM–80 µM. In this work, the composition used was POPC/RdLPS/PEGPE 55/25/20 (molar ratio) at a total lipid concentration of 100 µM. This corresponds to 55 µM of POPC (42 µg/mL), 15 µM PEG-PE (85 µg/mL) and 30 µM of RdLPS (80 µg/mL).

Lipid stock preparation
Lipids (POPC, PEGPE, RdLPS) were dissolved in chloroform at appropriate concentrations (5 mg/mL for POPC and RdLPS; 10 mg/mL for PEGPE) and stored in airtight glass vials with Teflon-lined caps at −20°C. Lipid stocks are stable for up to one month at −20°C in the dark.

Lipid-in-Oil Mixture Preparation
In a 7 mL glass vial, lipids are mixed in 200 μL hexadecane. Typically, 10 μL of POPC, 20 μL RdLPS and 12 μL PEG-PE were added and vortexed for 15 s. 1 mL long-chain mineral oil (Wako liquid paraffin) is added, and the mixture is heated for 1–2 h at 70°C, vortexed briefly to ensure homogeneity, and used directly.

CFE reaction Emulsification
In a microcentrifuge tube, 200 μL of the lipid-in-oil mixture was combined with 10 μL of the CFE reaction containing the desired components. The solution was emulsified by vortexing or rubbing on a rack, producing CFE droplets encapsulated in lipid micelles. The emulsion was transferred onto a new tube containing 250 μL of cationic solution and centrifuged at 5000 x g for 10 min to form liposomes.

RdLPS SCs Transfer and Phage Infection
Transfer of LPS liposomes: After centrifugation, the oil phase is removed. 10 µL of liposome pellet is transferred in a new 1.5 ml tube containing 90 µL of outer solution. Phage Addition and Imaging: Prepare 100 μL outer solution to which phage is added. Typically, 107–108 PFU/mL were added. In a 384-well plate, 90 μL of outer solution with phages was combined with 10 μL of liposomes. The plate was centrifuged at 600 x g for 5 min, followed by fluorescence microscopy imaging. Acquisition was done with an inverted microscope IX81 (Olympus) equipped with a camera and proper sets of filters. A 20x air objective or 40x and 100x oil objectives were used for observation. Image analysis was conducted with ImageJ and custom-made programs on Python.

Buffer compositions
Cationic solution: 270 mM K-glutamate, 25.5 mM Mg-glutamate, 45 mM Tris-HCl, autoclaved and adjusted to pH 8 and filter sterilized. Outer solution: 30 mM Maltodextrin, 90 mM K-glutamate, 4 mM Mg-glutamate, 1.5% PEG 8000, 4 mM amino acids, and an energy mix identical to the CFE reaction. The buffer is adjusted to pH 8 and filter sterilized.

CFE reaction and buffers
The CFE reactions used in this work were based on the CFE toolbox 2.056. The E. coli strain BL21-ΔrecBCD Rosetta 2, with a recBCD knockout to prevent the degradation of linear DNA57, was used for lysate preparation. Briefly, Cells were grown in 2x YT medium supplemented with phosphates, pelleted, washed, and lysed using a cell press. The lysate was centrifuged, and the supernatant was incubated at 37°C for 80 minutes. After a second centrifugation, the supernatant was dialyzed at 4°C for 3 h. Following a final centrifugation step, the lysate was aliquoted and stored at −80°C. The volume of the CFE reactions consisted of 33% cell extract, and 67% of the other components (energy mix, and amino acids). The reaction buffer included 50 mM Hepes (pH 8), 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.26 mM coenzyme A, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, 1 mM spermidine, 30 mM 3-phosphoglyceric acid (3-PGA), 1 mM dithiothreitol (DTT), 1.5% PEG 8000, and 20-40 mM maltodextrin. Amino acids were added at concentrations between 1.5 mM and 6 mM for each of the 20 amino acids (Supplementary Data 1). Magnesium (2–5 mM) and potassium (50-100 mM) concentrations were calibrated for deGFP synthesis and T7 phage production to obtain reproducible batches. Reactions were incubated at 30°C in either 1.5 mL tubes, 96-well plates, or encapsulated within liposomes in 384 well-plates. Fluorescence data from CFE reactions (Agilent Biotek H1m or Neo2) were treated with Microsoft PowerPoint or Kaleidagrah.

Determination of PFU per nL (PFU/nL)
For bulk cell-free reactions, PFU/nL titers were determined from 10 µL reactions. For liposome experiments, individual liposomes’ radii were measured under the microscope, and assuming spherical geometry, the volume of each liposome was calculated. The total encapsulated CFE reaction volume was then calculated by summing the volumes of all liposomes in the well. PFU counts were divided by this total volume, yielding PFU per nanoliter.

Microscopy data analysis
Microscopy images were acquired using an Olympus IX81 inverted microscope controlled by Metamorph software. Liposomes were imaged in 384-well plates, mounted on a custom-made microplate stage. Timelapse acquisitions across multiple wells were performed simultaneously. Temperature was maintained at 30°C by adjusting the microscope room temperature. Bright-field, green, and red fluorescence channels were recorded using phase contrast, GFP, and Texas Red filter sets. Image processing including segmentation and particle analysis was performed with ImageJ. exported data were plotted using Python scripts.

Figure 2A: Fluorescent images of RdLPS liposome populations encapsulating a dye were acquired at 20x magnification. Liposome segmentation and particle analysis were performed with ImageJ. Radius and volume measurements were used to plot size and volume distributions. The dataset includes 37,663 liposomes analyzed from three replicates. The mean liposome radius was 3.7 μm, with liposomes smaller than 20 μm in radius accounting for over 65% of the total encapsulated CFE reaction volume.

Figure 3D: RdLPS synthetic cells were infected by T7-mC-WT, T7-WT, T7-S* and T7-mC-S* phages, and their red fluorescence intensity was recorded after 13 hours of incubation with a microscope at a 20x magnification. For each condition and each replicate, the maximum fluorescence intensity inside each liposome characterized is measured. For the four conditions, a total of N = 28, 27, 28 and 31 liposomes respectively were characterized from three replicate.

Figure 3F: RdLPS synthetic cells encapsulating FITC-dextran were incubated with a serial dilution of T7-mC-S*. Liposome count was estimated by segmenting the green fluorescence channel using consistent thresholding across all datasets. Infected liposome count was determined by segmenting the red fluorescence channel after 15 h of incubation, using the same calibrated thresholding from a negative control (no phage). The multiplicity of infection (MOI) was calculated as the ratio of PFU added per well to the total liposome count. The infected liposome ratio was determined by dividing the red-channel liposome count by the total green-channel liposome count. Ratios were calculated from four replicate.

Figure 3G: RdLPS SCs either infected by T7-mC-S* phages or encapsulating mcherry expression gene cascade were incubated over 15 hours. Red fluorescence intensity was recorded with a microscope at 20x magnification. Using the procedure described in Supplementary text, the radius and concentration of in-situ synthesized mCherry were measured for N = 62 (mcherry gene encapsulation) and N = 36 (T7-mC-S* infection) liposomes. Liposomes were randomly selected from three replicate experiments in each condition. The linear fits slopes, intercept and R2 are (-0.76, 21.3, 0.35) for T7-mC-S* infection and (0.23, 22.1, 0.02) for mcherry gene encapsulation.

Figure 3I: RdLPS SCs either infected by T7-mC-S* phages or left uninfected had their green fluorescence intensity recorded for 15 hours every 15 minutes with a microscope at a 20x magnification. For each condition and each replicate, the timelapse images were cropped around isolated liposomes so that only one liposome would be present in each cropping series. The maximum intensity of each cropped image was then taken as the measurement of the green fluorescence intensity inside the liposome. For the two conditions, a total of N = 27 and 17 liposomes from three replicates respectively were characterized.

Figure 3K: Cluster count over time was analyzed using the green fluorescence channel. To isolate clusters, images from the timelapse green channel were processed by subtracting duplicates blurred time series (with different Gaussian sigma factors), effectively removing non-cluster fluorescence. The resulting cluster images were converted into masks, and clusters were counted over time using particle analysis. Identical parameters were applied to all conditions. Data represent clusters from 4 replicates. No clusters were detected in the absence of phage. Data were exported as CSV files and plotted using Python.

Figure 4A: To determine the typical timescale of DNA entry in liposomes, we recorded fluorescence intensity of SCs either encapsulating phage DNA or in presence of phage in the outer solution. For each of these two conditions, we considered two replicates observed simultaneously and obtained from the same initial liposome creation batches. Images were acquire every 15 minutes. For each replicate, the average fluorescence intensity of the 0.1% brightest pixels of each image was recorded and combined into the average over both replicates (master curve), then plotted as a function of time. A sigmoidal fit was then used to evaluate the initial and final plateau values of the master curve. The typical times of gene expression were evaluated from the time point where the master curve reaches halfway between the initial and final plateaus.

Sigmoid: f(x) = Ii - If / 1 + ek(t-t*) + If where Ii and If are respectively the initial and final plateau values of the master curve. We considered experimental points and not the sigmoid of best fit to evaluate t50%, which was found close to t*.

Figures 4B, D, E, G: In these experiments, liposomes encapsulated rhodamine-dextran (red fluorescence dye) and the T7 genome. Liposome count and radius were obtained through red-channel image segmentation, and liposome volume was calculated assuming spherical geometry. The total encapsulated CFE volume was estimated as the sum of all liposome volumes in a well. PFU production in liposomes was determined by liposome lysis and plating on an E. coli B lawn. PFU per nanoliter was estimated by dividing the recovered PFU by the total estimated liposome volume. Data represent s replicates for 4B, E, G and 4 replicates for 4D.

Figure 4H: A red fluorescent dextran dye and a phage holin gene circuit were co-encapsulated in liposomes with or without RdLPS. Red fluorescence intensity was recorded with a microscope at 20x magnification every 5 minutes for three hours. Leak ratios were obtained by superposing initial (average of the first 10 images colorized in red) and final (average of the last ten images artificially colorized green) images. Using the superpositions the liposomes were sorted into two groups red liposomes (leaked liposomes) and orange liposomes (no leak). After subtracting the fluorescence intensity of the background, the complement of the ratio of those differences was used as the leak ratio of each liposome. A leak ratio of 1 indicates that the entirety of the red fluorescent dextran dye leaked out of the liposome, while a leak ratio of 0 indicates that no dye has leaked from the liposome.

Leak ratio = 1 - (If - BGf) / (Ii - BGi) where Ii and If are the maximum fluorescence intensities inside the liposomes measured respectively right after creation and at the end of the incubation, while BGi and BGf the corresponding background fluorescence intensities. Over 50 liposomes were individually assessed from two replicate experiments.

Supplementary Fig. 35: Kinetic plots were generated in Python. mCherry fluorescence (T7-mC-S* infection) and Sybr Gold fluorescence data (from Fig. 3I and Supplementary Fig. 24) were analyzed, along with cluster formation data from Fig. 3K. Sigmoidal fits were applied to normalized mean Sybr Gold and mCherry fluorescence, while a Gaussian fit was used for normalized dCTP-FITC cluster counts. Min-max normalization was applied, and individual data points were included in Supplementary Fig. 35.

Statistics and reproducibility
For each experiment at least three independent replicates were made, except for experiments 4A and 4H (duplicate liposome batches). Plaque uncertainties were estimated at the time of counting (duplication or plaque belonging to the same spot). The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. For liposome analysis, liposomes were selected to be entirely contained within the imaging frame and have a well-defined outline that does not overlap with other liposomes. Liposomes were selected on a channel containing the volume marker and not the signal of interest. The micrographs shown in Figs. 2, 3 and 4 are representative images from one experiment. Each experiment was independently repeated three times with similar results.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The data (graphs, microscopy, movies, DNA sequences) generated and used in this study are available at the University of Minnesota Digital Conservancy (https://conservancy.umn.edu/) with the permanent URL: https://doi.org/10.13020/hmz3-yf4358, and under public available license, and in the supplementary file Supplementary Data 1. Source data are provided with this paper. The tree phage genomes generated in this study have been deposited in the European Nucleotide Archive (ENA) under the BioProject PRJEB104191. The genome of the three engineered phages T7-mC-S*, T7-GFP-S*, and T7-Split-S* are available under the accession codes OZ370878, OZ370879, OZ370880 respectively. Associated BioSample accessions are available at: T7-mC-S* (ERS27427136 [https://www.ebi.ac.uk/ena/browser/view/ERS27427136?dataType=SAMPLE]), T7-GFP-S* (ERS27427138 [https://www.ebi.ac.uk/ena/browser/view/ERS27427138?dataType=SAMPLE], and T7-Split-S* (ERS27427137 [https://www.ebi.ac.uk/ena/browser/view/ERS27427137?dataType=SAMPLE]). Source data are provided with this paper.

Code availability
The code and plotting scripts used in this study are available in the GitHub repository Noireauxlab-TXTL/A-synthetic-phage-cycle, archived on Zenodo under the DOI 10.5281/zenodo.1759335159.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors report phage t7 infection of liposomes containing E. coli cell-free extract. The liposomes and phage are cleverly modified to enable initiation of infection. The authors show by experiment gene expression w/in the liposomes due to the production of T7 RNA polymerase encoded w/in the infecting phage genome. The authors also show evidence of DNA replication and capsid assembly. Taken together these are significant results towards a fully synthetic T7 infection cycle. However, the title and abstract led me to expect more than what is being reported. E.g., the authors do not show infection of liposomes from liposome-produced T7 particles. I.e., multiple rounds of infection would be the strongest evidence of a complete phage infection cycle. There appear to be at least two barriers remaining between what the authors have demonstrated and a full infection cycle. (1) lysis, and (2) very low \"burst size\" (<1.0). Unfortunately, the manuscript is difficult to interpret in these two key aspects... E.g., the authors report the use of a holin expression cassette. It is not clear which ORF from T7 is being expressed. The sequence presented in Supplementary Spreadsheet (row 24) is not annotated. It's not totally clear that T7p14-T7holin-T7term -- ttaccagtgccaagaaggttccaggtacgtctcggatacccttactctgttgaaaacgaatagataggttaggcaactgaaacgattcggatcctgtattactattcttaactttatctgagaatagtcaatcttcggaaatcccaggtgtaatacgactcactatagggagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctctacaaataattttgtttaacaaccaaacaacttggttaacccgcacatacataaggaggtttttttatgctatcattagactttaacaacgaattgattaaggctgctccaattgttgggacgggtgtagcagatgttagtgctcgactgttctttgggttaagccttaacgaatggttctacgttgctgctatcgcctacacagtggttcagattggtgccaaggtagtcgataagatgattgactggaagaaagccaataaggagtgataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctattattgaccacttccgagtagaatcgtgcttcagtaagaccgtctcagaatcggccgtgaacaataaaatagtttcggttaataaaaggtcccgtctgaacttactgtgaattcgactaactgtcgcgtacactgtcagactcagacga -- encodes any ORFs of interest. Is this the right sequence? Also, T7 encodes three proteins involved in host cell lysis. Why are you only expressing a single gene for your synthetic lysis experiments? Which specific gene are you expressing? Next, given that the putative burst size from infected liposomes is less than 1.0 (Figure 4D).... how can you be 100% sure that you are not simply detecting viable phage left over from the initial infecting of liposomes (and not newly assembled phage). Next, infection dynamics appear to be ~100-fold slower than T7 infection of E. coli. Why? Finally, how is the genome getting into the liposome? Do you believe it to be mediated via transcription like during infection of E. coli? Why or why not? Further, please take care re: genome injection versus genome entry. E.g., F1A uses injection but it is not clear why there would be a syringe-like pressure driven mechanism for genome entry of liposomes when that is not observed during infection of E. coli. *** MINOR COMMENTS *** L162 refers to rate of infection but the data reported here is about the % of liposome infected, not rate. L109/110 — please clarify values and units (5.10-6 min-1; 3.10-9 min-138) F1b. Coli should be lower case c L31. \"It\" is in reference to what exactly? L66. No rRNA in the cell free? L168-9… how do you know extract concentration is the same in larger/smaller liposomes. Could such a difference explain what you are observing instead?", "reviewer_2": "The work by Levrier et al. describes a cell free expression system that replicates the majority of stages of the T7 phage cycle: adsorption, genome ejection, and phage replication. This was achieved by incorporating truncated RdLPS into liposomes composed of phospholipids (POPC and PE-PEG5000) encapsulating cell-free extract. The authors performed DOE to screen liposome compositions to form stable liposomes and successful phage transduction. The authors impressively characterize aspects of the system using fluorescence microscopy and a combination of clever labeling methods. Due to the confines of the CFE buffer conditions, the system is unable to replicate lysis and propagation without external intervention, and requires osmotic shock to release the phage. Though the results aren’t necessarily surprising, since it is known that LPS can induce genome ejection and that encapsulated CFE can express proteins, I believe the work is significant to a broad range of readers due to the growing interest of engineering biological systems from scratch, and ultimately building a fully synthetic cell. Additionally, this system is an interesting approach for studying viral infection because it offers a tractable bottom-up approach to study viral infection in a controlled environment, decoupling phage biology from the complexities of living bacteria. The manuscript is short and straightforward, and the data is intuitively presented. I believe the manuscript can be improved by expanding upon discussion and performing more in depth analysis taking advantage of their decoupled system. I would like to see the authors expand upon the discussion to include discussing implications of doing evolution experiments. What are advantages and some limitations. For example, I can envision being able to select for tail fiber mutants, exc., but due to having to input external intervention for release, would you also select for deletions in the lysis cassette, exc. Having to provide external intervention can also be an advantage, one can decouple ‘cheaters’ that more quickly replicate. I would also like the authors to expand discussion about broad utility of this approach. For example, could this system be used for T4, exc. Or is this system only limited to T7? And what do the authors envision for broadening the utility. Can the authors explain the discrepancy in timing compared to in vivo? T7 replication time in vivo is ~25 minutes, yet the authors observe T7 morphogenesis requiring ~9-10 hours. What if experiments were performed at 37oC? What phage and bacterial cells were used in the cited work. Please include this detail in text. Is this comparing apples to apples. I believe this suggests that the timing discrepancy isn’t due to adsorption.: ‘adsorption rate of an RdLPS-specific phage onto RdLPS SCs was estimated to be 5.10-6 min-1 109 (fig. S14), faster than reported values for bacterial cells (3.10-9 min)’ Can the authors put more emphasis on characterization, analysis, and discussion of phage burst size, and characterize at different temperatures: RT, 30, and 37oC. The authors very briefly mention burst size in the discussion as a potential bottleneck, but never characterize in detail. Can the authors be more quantitative in analysis of Figure 3D, and calculate the # of RFP per liposome. May give insight when comparing to PFUs? (This is just a suggestion to improve. These type of experiments can be challenging) I found it quite surprising that at high MOI (~3) the low % of liposomes that had mCherry expression. What is bottle neck? DNA delivery or phage expression? The authors can potentially answer this question through more detailed analysis of their data. Are the same % of liposomes showing fluorescence for DNA transduction, or is that a much higher %, indicating that the bottleneck is phage expression or assembly. I don’t fully agree with this interpretation of the data: ‘efficacy of genome ejection depends on the surface-to-volume ratio.’ Is it greater efficiency, or simply same number of fluorescent proteins, but small volume so more concentrated. I believe the data is potentially showing a concentration effect for expression, not ejection. Each liposome has the same number initial genomes, yet since the volume is smaller, the resulting expressed mCherry is more concentrated and adsorbs light more efficiently, thus emits higher fluorescence, or mCherry assembles more efficiently. The comparison to encapsulated plasmid is different, in that larger liposomes most likely encapsulate more plasmid, so the volume is larger, expresses more protein. Where in the authors’ system, each liposome presumably starts with 1 genome. Does dCTP-fitc inhibit RNA transcription. If so, why would the clusters dissipate? Could it be photobleaching? If it does inhibit, I would expect the phage not to assembly or package genome, thus not dissipate. Figure 3K is confusing. Not clear how disassembly is determined based on figure caption or text. Can the authors expand the histograms to show assembly and disassembly over the full time course, maybe overlapping, or on two different plots. Confusing how the data is shown as one histogram yet two different analyses. List time in figure for H and J. Why is the following expected, based on the timing of T7 Wt or previous CFE?: ‘DNA replication initiates at around 4–5 hours, consistent with the expected expression timing of T7 replication proteins’ The authors compare bulk versus SC expression, but do so at a single time point. Can the authors expand upon this to perform kinetic experiments for both, to ensure they are comparing apples to apples. In Figure 4, please show raw data with the fit data. How is PFU/nL calculated for liposomes. This information is important and isn’t included, or easily found. For the DNA concentration studies encapsulating T7 genomes (wt and mutants), what is the % of liposomes expressing mCherry as a function of DNA concentration, and how does this compare to T7 transduction in earlier studies. Could this give insight into what the bottleneck is? What is the red vertical line in Figure 4a? Why do authors think wt T7 was more efficient plus LPS. Regarding LPS inhibition of mutant T7. Was this caused by DNA release or irreversible phage binding? What if these experiments were performed at 37, 42, or even 50oC. The authors could quantify free DNA versus encapsulated DNA by adding DNase and comparing qPCR results, exc. Could dissipation be due to photobleaching: ‘Further, this approach enabled visualizing genome concatemer packaging 260 maturation into fluorescent phage capsids. Initially, green fluorescence clusters appeared inside 261 the liposomes within the characteristic time of DNA ejection and replication (fig. 4A, movie S5), 262 indicating fluorescent capsids localized near DNA concatemers (fig. 4E, fig. S31). Over time, the 263 clusters dispersed into smaller fluorescence foci, suggesting concatemer processing into progeny 264 phage particles.’ I believe the title and some of the claims are overreaching because lysis has to be induced. I think ‘transduction’ instead of infection is more accurate: This study demonstrates that LPS 323 alone is a sufficient membrane component for T7 genome internalization into SCs, providing 324 insights into the minimal requirements for phage infection. Please cite the work: ‘Unlike previous liposome studies with T5, which lacked cytoplasmic CFE’ I am confused by ‘low multiplicity of infection’. Doesn’t the system require high MOI: ‘The low multiplicity of infection 343 (MOI) constrains the number of productive infections, while DNA replication in CFE achieves 344 2–3-fold amplification28 compared to the 200–300-fold observed in bacterial hosts due to finite 345 resources in CFE.’", "reviewer_3": "This manuscript describes the use of cell free machinery in a membrane encapsulation to study phage replication. The presentation is excellent, with each step of the phage replication process carefully validated and analyzed. The experimental design is rigorous and described in a very detailed way, offering high novelty and significant potential for advancing both phage biology and phage engineering. The schematics are really helpful for understanding the experimental setup and interpreting results, although sometimes the explanations given in the figure legends are more clear than what is presented in the text. Overall, this work is important given the dearth of knowledge surrounding phage and their promise as novel therapeutics. Some minor clarifications, detailed below, would enhance the overall quality and significance of the work. 78-80: “to prevent unintended genome ejection” Was this observed or only hypothesized? 80: “RdLPS” Define this abbreviation; where possible, use fewer abbreviations throughout to enhance the flow and readability. 81: \"unlike wild-type T7 phage (T7 WT)\" Please clarify which type of LPS the T7 WT can interact with. 95-96: \"a chimeric protein (GFP-TF*) consisting of superfolder GFP fused to the N-terminal of the tip of the T7 RdLPS-specific tail fiber mutant (TF*)\" The description is confusing. Does GFP-TF* only contain the tip of the phage tail fiber or the whole mutant? 158: \"LPS were homogeneously distributed across liposomes, not all liposomes were infected \" Including the explanation in figure S8B – \"Only a fraction of RdLPS SCs binding GFP-TF* appear red, due to SCs leak, disruption or inactive SCs\" – would greatly clarify things here. 170: “intercalating fluorescent dye” Please specify. 174: \"these clusters dissipated as mCherry fluorescence increased\" The mCherry fluorescence is not consistent with the previous description of \"Upon the addition of T7-S* ….\" since the phage T7-S* shouldn't express mCherry during replication. Please clarify. 210: Consider describing TXTL for clarity. 229-230: \"At the lowest DNA concentration (10 pM), phage synthesis was more prevalent inside the SCs than in batch mode, likely due to favorable colocalization of gene expression machinery under confinement as compared to batch reactions\" Alternatively, the reason might be the colocalization of the phage components for assembly instead of the colocalization of gene expression machinery. 246-248: \"Next, to assess the effect of RdLPS incorporation on phage titer, we encapsulated T7-WT or T7-mC-S* genomes (10 pM) inside SCs with or without RdLPS into the membrane\" Why not use T7-WT and T7-S* for comparison? 288: Why are individual data points not shown in A and F? 298: Why was there a discrepancy in the number of replicates (3 vs 4)? 323-324: \"This study demonstrates that LPS alone is a sufficient membrane component for T7 genome internalization into SCs\" This claim might not be incorrect, but to be more rigorous, it would be better to use \"RdLPS\" and \"T7-S*\" specifically for this study. 344-345: \"DNA replication in CFE achieves 2–3-fold amplification\". Is this claim from another publication? It would be informative to show the DNA replication data in this study. 348: “substantial challenges” Please elaborate on this. 361-363: “Biocontainment” is not mentioned elsewhere in the manuscript. It seems a non sequitur to introduce it here. 435-436: \"custom-made programs on Python\" Please ensure the scripts are available. 453: What is the ratio between reaction buffer and cell extract? Or is the reaction buffer used for the cell lysis? Please clarify. 456-457: \"Amino acids were added at concentrations between 1.5 mM and 3 mM for each of the 20 amino acids.\" Please add a supplemental table for this. 500: \"Liposomes were randomly selected\" How was \"randomly\" achieved? 541: \"average of the last ten images colorized green\" Where does the green come from? 542-543: \"red liposomes (leaked liposomes) and orange liposomes (no leak)\" Why does the red refer to \"leaked liposomes\" and the orange to \"no leak\"? Figure 3G: Which MOI was used for this scatter plot? If the MOI were changed, would the result be different? Figure 4D: Change \"LPS\" to \"RdLPS\". Also, it seems the PFU of T7-WT surprisingly changed from RdLPS+ to RdLPS-. Any explanation? Figure S4C: \"membrane without LPS\" Change LPS to RdLPS. Figure S13C (right): Any explanation for the huge variation in mCherry signal?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to the reviewers’ comments\nReviewer #1 (Remarks to the Author):\nThe authors report phage t7 infection of liposomes containing E. coli cell-free extract. The liposomes and\nphage are cleverly modified to enable initiation of infection. The authors show by experiment gene\nexpression w/in the liposomes due to the production of T7 RNA polymerase encoded w/in the infecting\nphage genome. The authors also show evidence of DNA replication and capsid assembly.\nWe thank the Reviewer for their positive assessment. In addition to demonstrating gene expression, DNA\nreplication, and capsid assembly inside liposomes, we also show that viable phage particles are assembled\nwithin the synthetic cells from encapsulated genomes and can be released by osmotic shock.\nIn the revised manuscript, we also include a new experiment showing that infectious phages are produced\ninside synthetic cells after infection from phage particles initially added to the external solution. This new\nexperiment, presented in detail in response to another question and added to the revised manuscript (Fig.\n4G), demonstrates successful infection and a full replication cycle.\nTaken together these are significant results towards a fully synthetic T7 infection cycle. However, the title\nand abstract led me to expect more than what is being reported. E.g., the authors do not show infection of\nliposomes from liposome-produced T7 particles. I.e., multiple rounds of infection e be the strongest\nevidence of a complete phage infection cycle.\nWe thank the Reviewer for their comment. We agree that demonstrating multiple rounds of infection would\nprovide evidence of a self-sustaining synthetic phage infection cycle. Our work shows all the essential steps\nof the T7 cycle, both separately and in sequence. As mentioned in our response to the previous comment\nof the Reviewer, we now report the infection of synthetic cells from phages added to the outer solution, and\nthe recovery of newly assembled, viable phages from within infected synthetic cells following osmotic\nshock, similarly to the phageT7 cycles. While this establishes that phage-infected liposomes can produce\ninfectious T7 particles and release them through osmotic shock, it does not constitute an iterative cycle.\nOur in vitro phage infection cycle does not allow multiple rounds of propagation yet. We added the following\ntext to the second paragraph of the discussion to highlight the current limitations of the system (from Line\n380):\n‘Achieving a self-amplifying SC phage cycle with a greater burst size would require optimization of\nseveral critical steps, including DNA translocation efficacy, genome replication, minimization of phage\nrebinding and engineering of the phage genome to express an enzyme capable of lysing the LPS\nmembrane of the SCs. With such improvements, this system could be expanded to achieve multiple\ninfection cycles and evolutionary experiments.’\nThere appear to be at least two barriers remaining between what the authors have demonstrated and a full\ninfection cycle. (1) lysis, and (2) very low \"burst size\" (<1.0).\nWe agree that both lysis and low burst size are current limitations of the in vitro system.\n(1) Addressing active lysis (unlike osmotic shock which mimics the rupture of the bacterial outer membrane)\nwould likely require engineering a more complex synthetic cell membrane and introduction of a cognate\nlipase enzyme to the phage genome . As noted in the discussion and detailed in the question below, the\nmechanism of phage-induced lysis requires a non-isotonic system. In bacteria, this mechanical resistance\nis achieved by the cell wall - while phage lysis proteins only induce the burst by degrading the cell wall -\nturning the bacterial cell into a spheroplast (see lysis mechanism in the next answer). Our SCs, lacking a\ncell-wall equivalent, are hence analogous to a spheroplast by construction rendering gene encoded lysis\nineffective. While we agree that incorporating a synthetic cell wall to provide structural integrity and an inner\nmembrane (devoid of LPS) would be an important step toward enabling gene-encoded phage release, but\none that is beyond the scope of this study.\n(2) Regarding burst size, further improvements are needed in several areas to reach in vivo efficiency.\nThey include further enhancement of DNA translocation efficiency (in vitro we currently need around 100 phages\nto infect one SC while in vivo 1 phage for one bacterium suffices), improving DNA replication within the cell-\nfree system (1 genome yields 1-2 PFU in the CFE while in vivo 1 genome yields 50-300 PFU). One idea\ncould be better controlling the size of synthetic cells. In particular, smaller liposomes may support more\nefficient DNA translocation as noted by the surface-volume effect, replication and assembly, whereas the\nrelatively large size of liposomes used here likely limits overall productivity, but were essential to\ndemonstrate the different steps separately.\nIn conclusion, while we think that obtaining multiple rounds of infection is achievable by improving both\nmembrane and cell-free factors, achieving in vivo efficiency seems for now out of reach.\nWe added the following text to the second paragraph of the discussion to highlight the current limitations of\nthe system (from Line 380):\n‘Achieving a self-amplifying SC phage cycle with a greater burst size would require optimization of\nseveral critical steps, including DNA translocation efficacy, genome replication, minimization of\nphage rebinding and engineering of the phage genome to express an enzyme capable of lysing the\nLPS membrane of the SCs.’\nUnfortunately, the manuscript is difficult to interpret in these two key aspects...\nE.g., the authors report the use of a holin expression cassette. It is not clear which ORF from T7 is being\nexpressed. The sequence presented in Supplementary Spreadsheet (row 24) is not annotated. It's not\ntotally clear that T7p14-T7holin-T7term --\nttaccagtgccaagaaggttccaggtacgtctcggatacccttactctgttgaaaacgaatagataggttaggcaactgaaacgattcggat\ncctgtattactattcttaactttatctgagaatagtcaatcttcggaaatcccaggtg\nT7 promoter:\ntaatacgactcactataggg\nRBS:\nagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctctacaaataattttgtttaacaaccaaacaacttggtta\nacccgcacatacataaggaggttttttt\nHolin gene gene 17.5, lysis protein corresponding to T7 holin:\natgctatcattagactttaacaacgaattgattaaggctgctccaattgttgggacgggtgtagcagatgttagtgctcgactgttctttgggttaa\ngccttaacgaatggttctacgttgctgctatcgcctacacagtggttcagattggtgccaaggtagtcgataagatgattgactggaagaaag\nccaataaggagtgataa\nT7 terminator:\nctagcataaccccttggggcctctaaacgggtcttgaggggttttttgct\nTerminal spacer:\nattattgaccacttccgagtagaatcgtgcttcagtaagaccgtctcagaatcggccgtgaacaataaaatagtttcggttaataaaaggtcc\ncgtctgaacttactgtgaattcgactaactgtcgcgtacactgtcagactcagacga --\nencodes any ORFs of interest. Is this the right sequence? Also, T7 encodes three proteins involved in host\ncell lysis. Why are you only expressing a single gene for your synthetic lysis experiments? Which specific\ngene are you expressing?\nWe thank the Reviewer for raising this question. We now provide GenBank supplementary files with the\nannotated genes as supplementary information. Here is the annotation of the holin gene (ORF\ncorresponding to gene 17.5):\nInitial spacer:\nttaccagtgccaagaaggttccaggtacgtctcggatacccttactctgttgaaaacgaatagataggttaggcaactgaaacgattcggat\ncctgtattactattcttaactttatctgagaatagtcaatcttcggaaatcccaggtg\nT7 promoter:\ntaatacgactcactataggg\nRBS:\nagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctctacaaataattttgtttaacaaccaaacaacttggtta\nacccgcacatacataaggaggttttttt\nHolin gene gene 17.5, lysis protein corresponding to T7 holin:\natgctatcattagactttaacaacgaattgattaaggctgctccaattgttgggacgggtgtagcagatgttagtgctcgactgttctttgggttaa\ngccttaacgaatggttctacgttgctgctatcgcctacacagtggttcagattggtgccaaggtagtcgataagatgattgactggaagaaag\nccaataaggagtgataa\nT7 terminator:\nctagcataaccccttggggcctctaaacgggtcttgaggggttttttgct\nTerminal spacer:\nattattgaccacttccgagtagaatcgtgcttcagtaagaccgtctcagaatcggccgtgaacaataaaatagtttcggttaataaaaggtcc\ncgtctgaacttactgtgaattcgactaactgtcgcgtacactgtcagactcagacga\nThe T7 phage lysis cassette includes three main components: gene 17.5 (holin), gene 3.5 (lysozyme), and\ngene 17.7 (spanin). Each plays a distinct role in lysing the membrane and cell wall of the E. coli host. Holins\nform pores in the inner membrane, allowing the lysozyme to access the peptidoglycan layer in the\nperiplasm. The spanin transmembrane proteins help fusion of inner and outer membranes. Altogether,\nthese mechanisms weaken the host membrane and cell wall to enable lysis by osmotic pressure. In our\nsynthetic cell (SC) system, we express only the T7 holin gene (gene 17.5), which is the only component\nrelevant to our minimal membrane context.\nThis choice is based on both the structural and compositional differences between our SCs and natural\nbacterial cells. Our SCs consist of a single lipid bilayer decorated with LPS and do not contain a\npeptidoglycan cell wall, rendering lysozyme (which targets peptidoglycan) functionally irrelevant. Similarly,\nsince we lack both inner and outer membrane, the spanin complex is unnecessary. Furthermore, our data\nfrom Figure 4G suggest that LPS present on the SC membrane inhibits holin pore formation, consistent\nwith holins’ native action being limited to the inner membrane in E. coli. In natural infection, holins act to\ncreate a path for lysozyme delivery to the periplasm, but in our simplified system, that pathway and\ndownstream processes are not applicable. Thus, we focused solely on holin expression as it is the only\nmechanistically relevant lysis gene for synthetic cells composed of a single lipid bilayer. Our observations\nsupport the idea that the complete T7 lysis cassette is unnecessary to lyse our SCs, further underscoring\nthe need for external lysis.\nNext, given that the putative burst size from infected liposomes is less than 1.0 (Figure 4D).... how can you\nbe 100% sure that you are not simply detecting viable phage left over from the initial infecting of liposomes\n(and not newly assembled phage).\nIn the experiment shown in Figure 4D, we encapsulated T7 genomes inside SCs, rather than adding phage\nparticles in the outer solution. Rnase was added to the outer solution to degrade any residual or leaked\nexpression system, ensuring that no external phage gene expression could occur. Following expression\nand incubation, the SCs were lysed via osmotic shock, and the resulting viable phage particles were\nquantified. Under these conditions, all detected phages were assembled inside SCs from the encapsulated\ngenomes, confirming the internal synthesis of infectious phages.\nHowever, the point raised by the Reviewer becomes particularly relevant in experiments where phages are\nadded externally to infect SCs. In such cases, interpreting the total phage count after incubation is more\ncomplex, as it reflects a mixture of non-bound phages that remained in solution and newly assembled\nphages produced inside SCs. Currently, due to the low burst size (typically <1), the newly produced phage\ncount falls within the range of the background noise created by unbound input phage, making it challenging\nto confidently distinguish the two populations. To address this, we explored the development of tagged\nphages by incorporating detectable tags into capsid proteins expressed only inside SCs to enable selective\nidentification of newly produced phages. While technically promising, this strategy has so far proven difficult\nto implement consistently. We solved this issue by coming up with a new experiment as detailed in Figure\n4G.\nTwo populations of RdLPS SCs were prepared. One population encapsulates active CFE solution\n(without Rnase, called “active SCs”), the other population encapsulates CFE with Rnase A at 20 ug/mL\n(“dead SCs”), which prevents any T7 phage production in the liposomes. RdLPS SCs were incubated in\npresence of T7-mC-S* at an MOI of 100 for 2 hours at 30C during which phage adsorb and inject their\ngenomes into SCs. After 2 hours, a similar drop in titers was observed in the supernatants of the active (2\n±2x10^8 to 2 ±0.4x10^4 PFU/mL) and the dead SCs (3E ±2x10^8 to 2 ±0.2x10^4 PFU/mL) indicating\nsuccessful phage interaction with both populations of SCs. To remove as many unbound phages as\npossible, SCs were centrifuged and resuspended in a new feeding solution without phage and incubated\nfor 5 hours at 30C.\nAfter the incubation, SCs count was estimated in each replicate by taking a small volume fraction (5%) and\ncounted with the microscope. We obtained similar counts for the two populations: 323, 399 and 380 SCs\nfor the three active SCs replicates and 361, 342 and 456 for the dead SCs. The remaining SCs were\ncentrifuged, the supernatant discarded and lysed by addition of 100 uL of water. PFUs were recorded for\nboth populations. We observed 80 times more phages per liposome in the active compared to dead SCs .\nThis indicates that viable PFU are produced in SCs incubated with phages in the feeding.\nNew Fig. 4G:\nLines 300-304:\nTo determine the concentration of phages synthesized into the SCs when phages are added to\nthe outer solution, we applied an osmotic shock to an active and a dead SC populations. The\ndead SC population was created by adding Rnase A into the encapsulated CFE reaction, which\nensures that no phage is synthesized. We observed 80 times more phages per liposome in the\nactive compared to dead SCs.\nNext, infection dynamics appear to be ~100-fold slower than T7 infection of E. coli. Why?\nWe thank the Reviewer for raising this point. From the experiments presented in this first submission, it is\ndifficult to evaluate precisely this fold change, and our precedent analysis likely underestimated the kinetics.\nThere are several reasons for this.\n- From liposome infection experiments, mCherry fluorescence in T7mC-S infected SCs combines\nadsorption, DNA translocation, genome expression and mCherry maturation time. Moreover,\nmCherry expression characteristic time (t50%) does not correlate with phage packaging as\nmCherry gene expression could continue after genome packaging due to mCherry mRNA\nremaining in the SCs.\n- SYBR gold and dCTP data showing fluorescent cluster formation likely reflect concatemer\nformation (T7 DNA replication mechanism consists in the formation of concatemers of a dozen of\nT7 genomes in vivo.) While these clusters suggest DNA replication, T7 packaging could also occur\nconcurrently with T7 packaging. And in vivo, it is known that even after infection, approximately half\nof T7 genomes remain unpackaged.\n- Finally, our data on T7-Split-S expression suggest formation of packaged virions by adding a\nfluorescent reconstituted Split-GFP to the capsids but its characteristic time is also difficult to\nevaluate since it accounts for both Split-GFP reconstitution and could happen after the phage\nparticles are formed.\nOverall, our data presented in the first submission provided outer boundaries for the characteristic times\nbut clearly overestimated the infection kinetics. In particular, DNA translocation time and phage packaging\ntime in our SCs are overestimated. In order to improve our evaluation, we performed two additional\nexperiments.\nFor DNA translocation time estimation, we compared mCherry expression kinetics in SCs encapsulating\nT7mC-S genomes with SCs incubated in presence of T7mC-S in the outer solution. We estimated the\ntranslocation time by calculating the difference of t50% expression between these kinetics. In order to verify\nthis data, we engineered a new phage T7GFP-S containing a GFP reporter protein at the same loci as\nT7mCS using the same method as for T7mCS engineering. After purification of the T7GFPS genome and\npreparation of T7GFP phages, we performed the same experiment and evaluated the difference at t50%\nof gfp expression. Unsurprisingly we noticed that the t50% times were significantly shorter for GFP than for\nmCherry, coherent with shorter GFP maturation time as compared to Mcherry. We found similar differences\nfor the t50% of expression between encapsulated genome expression and the phage infection of about\n1.5h for T7-mC-S* and T7-GFP-S*.\nThese plots are now presented as new Supplementary Fig. S30 and the time values reported in amended\nfig 4A and presented in the manuscript (Lines 239-262, not pasted here for brevity).\nFig. S30:\nFor phage packaging time estimation, we recorded PFU every hour from either bulk cell-free T7 genome\nexpression of T7 genome at three different temperatures (24, 30 and 37C) and expression inside SCs at\n30C (Supplementary Figure S31). T7 genome concentration is set to 0.1 nM in presence of 0.1 mM dNTPs\nand 4% PEG in the CFE reaction. For the bulk T7 expression, the CFE reaction was stopped by addition\nof 10 times the volume in water on ice. PFU/nL were estimated by the plaque assay. For the SCs\nexperiment, RdLPS SCs encapsulating T7 at 0.1 nM were prepared with a red fluorescent dye as a volume\nmarker. Liposome count and radius were obtained through red-channel image segmentation, and liposome\nvolume was calculated assuming spherical geometry. The total encapsulated CFE volume was estimated\nas the sum of all liposome volumes in a well. PFU production at each timestep was determined by liposome\nlysis (the liposomes are sedimented, the outer solution is removed and 100 uL of water is added) and\nplating on an E. coli B lawn. PFU per nanoliter was estimated by dividing the recovered PFU by the total\nestimated liposome volume. Data represents 3 replicates.\nFig. S31:\nThe data obtained indicates that T7 packaging depends on temperature, the greater the temperature the\nfaster the packaging. After one hour between 10^2-10^3 PFU/nL are obtained at 30C and 37C while no\nPFU are detected at 24C. Interestingly, similar titers are obtained between bulk and SCs packaging at 30C.\nAfter 5 hours of incubation, we find titers in this range with 10±2x10^4 PFU/nL, 7±3x10^4 PFU/nL, 2±1x10^4\nPFU/nL in bulk CFE at 24C, 30C and 37C and 9±3x10^4 PFU/nL in SCs at 30C. Given that at 0.1 nM if all\ngenomes initially added to the CFE reaction become a PFU in absence of DNA replication 6x10^4 PFU/nL\nare expected. We hence obtain slightly higher titer values than expected suggesting a DNA replication\nfactor of about 1.5 in SCs at 30C. This seemingly small DNA replication to put in perspective since it is hard\nto evaluate genome integrity while only viable phage particles are counted with the agar overlay method.\nOverall, we conclude that phage packaging time starts within the first hour. After one hour, 1-2% of the final\ntiter is attained at 30C in bulk and in SCs and reaches >50% of final titer after 2 hours of incubation, while\ncontinuing to increase until 5 hours of incubation. We hence chose a characteristic packaging time of 1-2\nhours is our SC model at 30C.\nTaken together, these two new experiments considerably shorten the infection dynamics of our SC model.\nAdsorption mainly happens in the first 10 minutes, DNA translocation in about 1.5 hours and phage\npackaging in about 1-2 hours. We can hence estimate our synthetic infection cycle of about 3 hours at 30C.\nConsidering 30 minutes for the duration of T7 phage cycle at 30C, we obtain a 6-fold and not a 100-fold.\nThe above data is presented in the main text , Lines 239-262, not copied here to reduce redundancy).\nNonetheless, we agree that the extended timeline for the T7 cycle in our system compared to the ~30-\nminute replication cycle observed in vivo requires further discussion. To try to explain these differences,\nour minimal model system is far from the complex bacteria, probably less efficient both at the membrane\nas well as the cytoplasmic level. Several factors likely contribute to this difference:\n1. First, DNA transduction into liposomes is undoubtedly less efficient than natural infection (about 10\nminutes in an E. coli bacterium at 30C was previously reported), due to the simplified membrane\narchitecture; the LPS-coated bilayer lacks additional E. coli outer membrane components that would\nfacilitate faster genome entry.\n2. Second, a cell-free expression system is a diluted cytoplasm that achieves slower transcription and\ntranslation kinetics compared to in vivo. Taking 20 minutes for the expression, DNA replication and\npackaging time in vivo, our system is approximately 5 times slower than in vivo. It is remarkable given\nthat E. coli cytoplasm in an active CFE reaction is diluted about 25 times.\nThe latter explanation was not incorporated into the main text due to space limitations.\nFinally, how is the genome getting into the liposome? Do you believe it to be mediated via transcription like\nduring infection of E. coli? Why or why not? Further, please take care re: genome injection versus genome\nentry. E.g., F1A uses injection but it is not clear why there would be a syringe-like pressure driven\nmechanism for genome entry of liposomes when that is not observed during infection of E. coli.\nWe thank the reviewer for this important fundamental question. As we do not have a definite answer for the\nexact mechanism in play in our system and due to contradiction in literature, we decided to follow the\nreviewer suggestion and downplay the claim by systematically replacing ‘genome injection’ with ‘genome\nentry’ throughout the manuscript. A structural analysis would be required to assess this question correctly\nand we believe it is outside of the scope of this work.\n*** MINOR COMMENTS ***\nL162 refers to rate of infection but the data reported here is about the % of liposome infected, not rate.\nWe thank the reviewer for raising this point. It is indeed a mistake that is now corrected in our revised\nmanuscript as follows (Line 166): “The efficacy of infection depended on the multiplicity of infection (MOI),\ndefined as the ratio of plaque-forming units (PFU) to SCs.”\nL109/110 — please clarify values and units (5.10-6 min-1; 3.10-9 min-138)\nAs noted by another reviewer, the units and the comparison are not appropriate as it involved different\nphage-host systems and experimental conditions, making it difficult to draw meaningful conclusions. Ideally,\nwe would compare the adsorption rate of T7 phages onto bacterial cells from which the LPS used in our\nsynthetic cells was purified, under identical conditions and MOI. However, the RdLPS-expressing strain\nused in prior work is a proprietary Sigma strain and was not available to us, so we relied on literature\nestimates for context.\nThat being said, in our own system, we observe that approximately 93% of phages are bound within the\nfirst 5 minutes at an MOI of 1, suggesting efficient initial adsorption. To avoid overinterpretation and better\nreflect our data, we revised the text to remove the rate comparison and instead report our empirical\nobservation as follows (Lines 112-114): “At an initial MOI of 1, where binding sites are in excess, over 90%\nof phages bind within the first 5 minutes, suggesting efficient initial adsorption.”\nWe also improved the corresponding exponential decay fit in the Supplementary Fig. S14, as it did not\naccurately represent the data:\nF1b. Coli should be lower case c\nThank you. This was corrected in the main Figure 1.\n“It” refers to cell-free gene expression systems. We made it clearer as follows: “Cell-free gene expression\noffers a testbed for validating and enriching our understanding of life’s fundamental principles and\nconstructing synthetic life systems”.\nL66. No rRNA in the cell free?\nWe thank the Reviewer for noticing this point. There is indeed rRNA (ribosomes) in the cell-free reaction.\nWe made the caption of Figure 1 clearer as follows:\n“A cell-free expression (CFE) system devoid of DNA, messenger RNA, and phage DNA and\nproteins is encapsulated into the liposome to allow for phage genome expression upon DNA\nejection, initiating replication and virion production.”\nL168-9… how do you know extract concentration is the same in larger/smaller liposomes. Could such a\ndifference explain what you are observing instead?\nWe thank the Reviewer for this important observation. Our SCs are prepared by the inverted emulsion\nmethod. The variation in the concentrations of encapsulated components as a function of liposome radius\nstems from the emulsion step determined by Poisson law. However, at our liposome and concentration\nscales, we believe this effect is negligible for extract components (uM range) except for components\nencapsulated at very low concentrations such as DNA in the nanomolar range.\nTo assess whether extract concentration varies across liposomes of different sizes, we measured both\nmCherry fluorescence intensity and liposome radius, allowing us to estimate mCherry concentration\nindependently of volume (Supplementary Fig. S20). In encapsulated DNA experiments, we observed that\nmCherry concentration remains constant across liposome sizes, indicating that the cell-free reaction and\nDNA are uniformly distributed during encapsulation, and that there is no detectable volume-dependent\ndilution.\nHowever, in the case of phage infection, the outcome differs: smaller liposomes tend to show higher\nmCherry concentration. We hypothesize that this difference arises from a surface-to-volume effect, as\nphage infection and DNA injection are initiated at the membrane. Smaller liposomes have a higher surface-\nto-volume ratio, which may increase the likelihood of successful infection and genome delivery relative to\nlarger liposomes. This supports the idea that membrane-localized processes, rather than gene expression\nefficiency differences, are responsible for the observed effect.\nTo further study this effect, we performed a more complete analysis of mCherry concentration in SCs as a\nfunction of radius across MOI values (3000, 300, 30 and 3). We observe the same trend, with smaller MOI\nfavoring smaller liposomes while high MOI saturates the liposomes with phage genomes (new\nSupplementary Fig. S20). This suggests that the effect is due to the phage concentration and not by SCs\nextract stochasticity of encapsulation.\nThis paragraph was re-written accordingly (Lines 171-176). Fig. S20:\n8\nThe work by Levrier et al. describes a cell free expression system that replicates the majority of stages of\nthe T7 phage cycle: adsorption, genome ejection, and phage replication. This was achieved by\nincorporating truncated RdLPS into liposomes composed of phospholipids (POPC and PE-PEG5000)\nencapsulating cell-free extract. The authors performed DOE to screen liposome compositions to form\nstable liposomes and successful phage transduction. The authors impressively characterize aspects of the system\nusing fluorescence microscopy and a combination of clever labeling methods. Due to the confines of the\nCFE buffer conditions, the system is unable to replicate lysis and propagation without external intervention\nand requires osmotic shock to release the phage. Though the results aren’t necessarily surprising, since it\nis known that LPS can induce genome ejection and that encapsulated CFE can express proteins, I believe\nthe work is significant to a broad range of readers due to the growing interest of engineering biological\nsystems from scratch and ultimately building a fully synthetic cell. Additionally, this system is an interesting\napproach for studying viral infection because it offers a tractable bottom-up approach to study viral infection\nin a controlled environment, decoupling phage biology from the complexities of living bacteria.\nThe manuscript is short and straightforward, and the data is intuitively presented. I believe the manuscript\ncan be improved by expanding upon discussion and performing more in-depth analysis taking advantage\nof their decoupled system.\nWe thank the Reviewer for the positive assessment.\nI would like to see the authors expand upon the discussion to include discussing implications of doing\nevolution experiments. What are advantages and some limitations. For example, I can envision being able\nto select for tail fiber mutants, exc., but due to having to input external intervention for release, would you\nalso select for deletions in the lysis cassette, exc. Having to provide external intervention can also be an\nadvantage, one can decouple ‘cheaters’ that more quickly replicate.\nThank you for this suggestion. We added the following text to the second paragraph of the discussion (Lines\n383-388):\n“...this system could be expanded to achieve multiple infection cycles and evolutionary\nexperiments. While external intervention for phage release may limit selection on the lysis\ncassette, potentially leading to its deletion, but also offers a unique advantage by allowing\nexperimental control over phage population dynamics, including the suppression of fast replicating\n'cheaters.' This setup could enable targeted selection for traits at the membrane such as tail fiber\nmutations, or in the cytoplasm such as defense systems, facilitating directed evolution of host\nspecificity.”\nI would also like the authors to expand discussion about broad utility of this approach. For example, could\nthis system be used for T4, exc. Or is this system only limited to T7? And what do the authors envision for\nbroadening the utility.\nWe thank the Reviewer for this suggestion. We have added a paragraph to the discussion addressing the\nbroader utility of our approach (Lines 373-379):\n“While our current system is optimized for T7, which uses only LPS as its receptor, the strategy could be\nextended to phages like T4 that require both LPS and specific outer membrane protein receptors. To\naccommodate such phages, the required protein receptor could be incorporated into the outer membrane\neither by direct expression within the liposomes, allowing spontaneous membrane insertion, or via recently\nreported secretion systems capable of inserting membrane proteins( Matsubayashi, Hideaki, Yutetsu\nKuruma, and Takuya Ueda. \"In vitro synthesis of the E. coli Sec translocon from DNA.\" Angewandte Chemie\n126.29 (2014): 7665-7668.). The T7-based platform reported here is a foundational step toward a more\ngeneralizable in vitro system for studying diverse phage infections.”\nCan the authors explain the discrepancy in timing compared to in vivo? T7 replication time in vivo is ~25\nminutes, yet the authors observe T7 morphogenesis requiring ~9-10 hours. What if experiments were\nperformed at 37oC?\nWe thank the Reviewer for raising this point. From the experiments presented in the first submission, it is\ndifficult to evaluate precisely this fold change, and our precedent analysis likely underestimated the kinetics.\nThere are several reasons for this, temperature is one of these but not a substantial one as shown by the\nnew data presented here.\n- From liposome infection experiments, mCherry fluorescence in T7mC-S infected SCs combines\nadsorption, DNA translocation, genome expression and mCherry maturation time. Moreover,\n10\nmCherry expression characteristic time (t50%) does not necessarily correlates with phage\npackaging as mCherry gene expression could continue after genome packaging due to remaining\nmCherry mRNA remaining in the SCs.\n- SYBR gold and dCTP data showing fluorescent cluster formation likely reflect concatemer\nformation (T7 DNA replication mechanism consist in the formation of concatemers made of a dozen\nof T7 genomes in vivo.) While these clusters suggest DNA replication, DNA replication could also\noccur concurrently with T7 packaging. And in vivo, it is known that even after infection,\napproximately half of T7 genomes remain unpackaged.\n- Finally, our data on T7-Split-S expression suggest the formation of packaged virions by adding a\nfluorescent reconstituted Split-GFP to the capsids but its characteristic time is also difficult to\nevaluate since it accounts for both Split reconstitution and could happen after the phage particles\nare formed.\nOverall, our data presented previously provided outer boundaries for the characteristic times but likely\noverestimated the infection kinetics. In particular, DNA translocation time and phage packaging time in the\nSCs are likely overestimated. In order to improve our evaluation, we performed two additional experiments.\nAs suggested, to investigate temperature effects, we performed a kinetic analysis of T7 production (in batch\nmode cell-free reactions) at room temperature, 30°C, and 37°C, and observed a slight acceleration of phage\nproduction with increasing temperature (see results below). Live imaging at 37°C is not feasible with our\ncurrent microscopy setup due to issues with thermal stability and evaporation, which required us to maintain\nconditions at 30°C without shaking to preserve liposome positioning. Note, however, that 30C seems to be\nan optimum for this system.\nFor DNA translocation time estimation, we compared mCherry expression kinetics in SCs encapsulating\nT7mC-S genomes with SCs incubated in presence of T7mC-S in the outer solution. We estimated the\ntranslocation time by calculating the difference of t50% expression between these kinetics. In order to verify\nthis data, we engineered a new phage T7GFP-S containing a GFP reporter protein at the same loci as\nT7mCS using the same method as for T7mCS engineering. After purification of the T7GFPS genome and\npreparation of T7GFP phages, we performed the same experiment and evaluated the difference at t50%\nof GFP expression. As expected, we noticed that the t50% times were significantly shorter for GFP than for\nmCherry coherent with shorter GFP maturation time as compared to mCherry. We found similar differences\nbetween the t50% of expression of encapsulated genome expression and the phage infection of about 1.5h\nfor T7-mC-S* and T7-GFP-S*.\nThese plots are now presented as Supplementary new Fig. S30 and the time values reported in amended\nfig 4A and presented in the manuscript (Lines 239-262, not pasted here for brevity).\nFig. S30:\nexpression of T7 genome at three different temperatures (24, 30 and 37C) and expression inside SCs at\n30C (Supplementary Fig. S31). T7 genome concentration is set at 0.1 nM in presence of 0.1 mM dNTPs\nand 4% PEG in the CFE reaction. For the bulk T7 expression, the CFE reaction was stopped by addition\nof 10 times the volume in water on ice. PFU/nL were estimated by plaque assay. For the SCs experiment,\nRdLPS SCs encapsulating T7 at 0.1 nM were prepared with a red fluorescent dye as a volume marker.\nLiposome count and radius were obtained through red-channel image segmentation, and liposome volume\nwas calculated assuming spherical geometry. The total encapsulated CFE volume was estimated as the\nsum of all liposome volumes in a well. PFU production at each timestep was determined by liposome lysis\n(the liposomes are sedimented, the outer solution is removed and 100 uL of water is added) and plating on\nan E. coli B lawn. PFU per nanoliter was estimated by dividing the recovered PFU by the total estimated\nliposome volume. Data represents 3 replicates.\ntemperatures. After one hour between 10^2-10^3 PFU/nL are obtained at 30C and 37C while no PFU are\ndetected at 24C. Interestingly, similar titers are obtained between bulk and SCs packaging at 30C. After 5\nhours of incubation, we find titers in this range with 10±2x10^4 PFU/nL, 7±3x10^4 PFU/nL, 2±1x10^4\nPFU/nL in bulk CFE at 24C, 30C and 37C and 9±3x10^4 PFU/nL in SCs at 30C. Given that at 0.1 nM if all\ngenomes initially added to the CFE reaction become a PFU in absence of DNA replication 6x10^4 PFU/nL\nare expected. Hence, we obtain slightly higher titer values than expected suggesting a DNA replication\nfactor of about 1.5 in SCs at 30C. This seemingly small DNA replication to put in perspective since it is hard\nto evaluate genome integrity while only viable phage particles are counted with the agar overlay method.\nOverall, we conclude that phage packaging time starts within the first hour. After one hour, 1-2% of the final\ntiter is attained at 30C in bulk and in SCs and reaches >50% of final titer after 2 hours of incubation, while\ncontinuing to increase until 5 hours of incubation. Hence, the characteristic packaging time is on the order\nof 1-2 hours in our SC model at 30C.\nTaken together, these two new experiments show a much faster infection dynamics of our SC model.\nAdsorption mainly happens in the first 10 minutes, DNA translocation in about 1-2 hours and phage\npackaging in about 1-2 hours. The synthetic infection cycle is on the order of 3 hours at 30C. Considering\n30 minutes for the duration of T7 phage cycle at 30C in vivo, we obtain a 6-fold and not a 100-fold.\nNonetheless, we agree that the extended timeline for the T7 cycle in our system compared to the ~30-\nminute replication cycle observed in vivo requires further discussion. To try to explain these differences,\nour minimal model system is far from the complex bacteria, probably less efficient both at the membrane\nas well as the cytoplasmic level. Several factors likely contribute to this difference:\n1. First, DNA transduction into liposomes is undoubtedly less efficient than natural infection (about 10\nminutes in an E.coli bacterium at 30C was previously reported), due to the simplified membrane\narchitecture; the LPS-coated bilayer lacks additional E. coli outer membrane components that would\nfacilitate faster genome delivery.\n2. Second, a cell-free expression system is a diluted cytoplasm that achieves slower transcription and\ntranslation kinetics compared to in vivo. Taking 20 minutes for the expression, DNA replication and\npackaging time in vivo, our system is approximately 5 times slower than in vivo. This is remarkable given\nthat E. coli cytoplasm in an active CFE reaction is diluted about 25 times.\nThe latter explanation was not incorporated into the main text due to space limitations.\ncomparing apples to apples. I believe this suggests that the timing discrepancy isn’t due to adsorption.:\n‘adsorption rate of an RdLPS-specific phage onto RdLPS SCs was estimated to be 5.10-6 min-1 109 (fig.\nS14), faster than reported values for bacterial cells (3.10-9 min)’\nWe thank the Reviewer for this comment. We agree that the original comparison was not appropriate, as it\ninvolved different phage-host systems and experimental conditions, making it difficult to draw meaningful\nconclusions. Ideally, we would compare the adsorption rate of the T7 phage onto bacterial cells from which\nthe LPS used in our synthetic cells was purified, under identical conditions and MOI. However, the RdLPS-\nexpressing strain used in prior work is a proprietary Sigma strain and was not available to us, so we relied\non literature estimates for context.\nThat being said, in our own system, we observe that approximately 93% of phages are bound within the\nfirst 5 minutes at an MOI of 1, suggesting efficient initial adsorption. To avoid overinterpretation and better\nreflect our data, we revised the text to remove the rate comparison and instead report our empirical\nobservation as follows (Lines 112-114): “At an initial MOI of 1, where binding sites are in excess, over 90%\nof phages bind within the first 5 minutes, suggesting efficient initial adsorption.”\nWe also improved the corresponding exponential decay fit in the Supplementary Fig. S14, as it did not\naccurately represent the data:\nCan the authors put more emphasis on characterization, analysis, and discussion of phage burst size, and\ncharacterize at different temperatures: RT, 30, and 37oC. The authors very briefly mention burst size in the\ndiscussion as a potential bottleneck, but never characterize in detail.\nThank you for this valuable suggestion. In response, as already presented above, we included additional\ndata to better characterize the kinetics of phage production under different temperature conditions.\nSpecifically, we performed a new experiment in which purified T7 genomes were expressed in bulk cell-\nfree reactions at 24C, 30C, and 37C (already presented above, Supplementary Fig. S31). Plaque-forming\nunits (PFUs) were measured every hour for 5 hours. These results show that temperature has a strong\neffect on the kinetics of phage production, with faster accumulation of viable phage particles at higher\ntemperatures. However, the final yields remain similar across temperatures except for 37C that has a\nslightly smaller yield (3x less) compared to 24 and 30C. For burst size, we observe that the DNA replication\nfactor (here ~1.5 fold for SCs at 30C) limits the achievable burst size when SCs are infected from the\noutside. These data are now included in Supplementary Figure S31:\nencapsulated T7 genomes inside SCs, rather than adding phage particles in the outer solution. Rnase was\nadded to the outer solution to degrade any residual or leaked expression system, ensuring that no external\nphage gene expression could occur. Following expression and incubation, the SCs were lysed via osmotic\nshock, and the resulting viable phage particles were quantified. Under these conditions, all detected phages\nwere assembled inside SCs from the encapsulated genomes, confirming the internal synthesis of infectious\nphages.\nHowever, we did not characterize the burst size in experiments where phages are added externally to infect\nSCs because interpreting the total phage count after incubation is more complex, as it reflects a mixture of\nnon-bound phages that remained in solution and newly assembled phages produced inside SCs. Currently,\ndue to the low expected burst size (typically <1), the newly produced phage count falls within the range of\nthe background noise created by unbound input phage, making it challenging to confidently distinguish the\ntwo populations.\nTo address this, we explored the development of tagged\nphages by incorporating detectable tags into capsid proteins expressed only inside SCs to enable selective\nidentification of newly produced phages.\nWhile technically promising, this strategy has so far proven difficult to implement consistently. We solved\nthis issue by coming up with a new experiment detailed in Figure 4G.\nTwo populations of RdLPS SCs were prepared. One population encapsulates an active CFE solution\n(without Rnase, called “active SCs”), the other population encapsulates CFE with Rnase A at 20 ug/mL\n(“dead SCs”), which prevents any T7 phage production into the liposomes. RdLPS SCs were incubated in\npresence of T7-mC-S* at an MOI of 100 for 2 hours at 30C during which phages adsorb and inject their\ngenomes into SCs. After 2 hours, a similar drop in titers was observed in the supernatants of the active (2\n±2x10^8 to 2 ±0.4x10^4 PFU/mL) and the dead SCs (3E ±2x10^8 to 2 ±0.2x10^4 PFU/mL) indicating\nsuccessful phage interaction with both populations of SCs. To remove as many unbound phages as\npossible, SCs were centrifuged and resuspended in a new feeding solution without phage and incubated\nfor 5 hours at 30C. After the incubation, SCs count was estimated in each replicate by taking a small volume\nfraction (5%) and counted with the microscope. We obtained similar counts for the two populations: 323,\n399 and 380 SCs for the three active SCs replicates and 361, 342 and 456 for the dead SCs. The remaining\nSCs were centrifuged, the supernatant discarded and lysed by addition of 100 uL of water. PFUs were\n15\nrecorded for both populations. We observed 81 times more phages per liposome in the active compared to\ndead SCs. This indicates that viable PFU are produced in SCs incubated with phages in the feeding.\nNew Fig. 4G:\nCan the authors be more quantitative in analysis of Figure 3D, and calculate the # of RFP per liposome.\nMay give insight when comparing to PFUs? (This is just a suggestion to improve. These types of\nexperiments can be challenging)\nWe thank the reviewer for this suggestion that would certainly add more information to the study. However,\nthis is a challenging experiment that requires substantial work, beyond what we could do in the premise of\nthis revision, as we prioritized other experiments, as suggested by this reviewer and the others.\nI found it quite surprising that at high MOI (~3) the low % of liposomes that had mCherry expression. What\nis bottle neck? DNA delivery or phage expression? The authors can potentially answer this question through\na more detailed analysis of their data. Are the same % of liposomes showing fluorescence for DNA\ntransduction, or is that a much higher %, indicating that the bottleneck is phage expression or assembly.\nWe assume the reviewer was referring to low rather than high MOC (~3). This is Indeed intriguing. Yet,\nthere are multiple variables to consider from which we cannot devise a clear-cut answer. These could\ninclude the low concentration of small number or a single genome within the liposome that renders\nreplication, transcription and/or translation significantly less effective, compounded by variability of\nliposome volume (significantly higher than a bacterial cell for most liposomes), encapsulation efficiency and\nLPS distribution. The lack of cellular regulation and homeostasis in the non-living liposome together with\nexpected non-linearity of genome expression: assume 1st genome internalized, T7RNAP expressed →\nmCherry expressed. Now a second genome internalized: T7RNAP expressed that makes more\ntranscription → disbalanced transcription/translation → nonlinearity of mCherry production versus #\ngenome internalized. Taken together, we considered that such discussion is best left out of this manuscript.\nI don’t fully agree with this interpretation of the data: ‘efficacy of genome ejection depends on the surface-\nto-volume ratio.’\nIs it greater efficiency, or simply same number of fluorescent proteins, but small volume so more\nconcentrated. I believe the data is potentially showing a concentration effect for expression, not ejection.\nEach liposome has the same number initial genomes, yet since the volume is smaller, the resulting\nexpressed mCherry is more concentrated and adsorbs light more efficiently, thus emits higher fluorescence,\nor mCherry assembles more efficiently. The comparison to encapsulated plasmid is different, in that larger\nliposomes most likely encapsulate more plasmid, so the volume is larger, expresses more protein. Where\nin the authors’ system, each liposome presumably starts with 1 genome.\nTo assess whether mCherry concentration varies across liposomes of different sizes, we measured both\nmCherry fluorescence intensity and corresponding liposome radius, allowing us to estimate mCherry\nconcentration independently of volume. In encapsulated DNA experiments, we observed that mCherry\n16\nconcentration remains constant across liposome sizes, indicating that the CFE reaction and DNA are\nuniformly distributed during encapsulation, and that there is no detectable volume-dependent dilution effect.\nIn the plasmid encapsulation experiment, larger liposomes have more plasmids but identical plasmid\nconcentration as smaller ones if we neglect the noise associated with the encapsulation process due to the\nrelatively low DNA concentration (~10 nM).\nHowever, in the case of phage infection, the outcome differs: smaller liposomes tend to show higher\nmCherry concentration. We hypothesize that this difference arises from a surface-to-volume effect, as\nphage infection and DNA injection are initiated at the membrane. Smaller liposomes have a higher surface-\nto-volume ratio, which may increase the likelihood of successful infection and genome delivery relative to\nlarger liposomes. This supports the idea that membrane-localized processes, rather than gene expression\nefficiency differences, are responsible for the observed effect.\nTo further study this effect, we performed a more complete analysis of mCherry concentration in SCs as a\nfunction of radius across MOI values (3000, 300, 30 and 3) (Supplementary Fig. S20). We observe the\nsame trend, with smaller MOI favoring smaller liposomes while high MOI saturates the liposomes with\nphage genomes.\nThe relevant paragraph was re-written accordingly (Lines 171-176). Fig. S20:\nDoes dCTP-fitc inhibit RNA transcription. If so, why would the clusters dissipate? Could it be\nphotobleaching? If it does inhibit, I would expect the phage not to assembly or package genome, thus not\ndissipate.\nWe thank the reviewer for this question.\nTo bring an element of answer we did an additional experiment (Supplementary Fig. S23). A gene fragment\nof about 1 kbp containing a mcherry reporter gene under a T7 promoter (T7-mcherry) was prepared by\nPCR adding dNTPs and 10% of FITC-dCTP. After PCR and PCR purification, the gene fragment FITC-\ndCTP incorporation was quantified by calibration with pure FITC-dCTP. We found that each fragment had\non average 14 dCTP-FITC per dsDNA reporter fragment (1.4% incorporation).\nmcherry). While we observe that both kinetics and final fluorescence values are impacted (<15%) by the\nincorporation of FITC-dCTP, it does not inhibit transcription in our CFE reaction.\nMoreover, we argue that photobleaching is not the reason why dCTP-FITC clusters dissipate. By having a\ncloser look at the timelapse movies, (as exemplified below, green fluorescence channel), we observe SCs\nwith cluster formation and disassembly (white arrows). Between frames in the middle and on right, we see\ncluster form and disassemble but not at the same time. This variability in cluster formation and disassembly\ntime excludes photobleaching as the main cause for disassembly.\nof DNA concatemers. Another cause could be hydrolyzing FITC-dCTP into dCTP. However, the T7 phage\ndoes have its own dNTP metabolism (unlike phage T4).\nThis is now referred to in the main text (Lines 191-192):\nWe controlled for proper transcription in presence of the fluorescent nucleotide and discarded\npotential photobleaching (Fig. S23).\nFigure 3K is confusing. Not clear how disassembly is determined based on figure caption or text. Can the\nauthors expand the histograms to show assembly and disassembly over the full-time course, maybe\noverlapping, or on two different plots. Confusing how the data is shown as one histogram yet two different\nanalyses.\nWe thank the Reviewer for sharing about their confusion around Figure 3K. This plot represents the count\nof clusters over time (from four different replicates) and does not consist in aggregation of two histograms.\nWe agree that the original representation using a histogram to infer both assembly and disassembly\ndomains based on a Gaussian fit could lead to misinterpretation: the apparent bimodal shape of the\ndistribution likely results from variability across the four replicate experiments, making the boundaries of\nthe two domains problematic.\nWe revised the figure to improve clarity. Specifically, we removed the predefined “assembly” and\n“disassembly” time domains and instead added directional arrows to indicate the overall trends in assembly\nand disassembly over time. This allows the reader to see the temporal progression more intuitively without\nrelying on ambiguous histogram boundaries.\nList time in figure for H and J.\nTimes are now included in Figures 3H and 3J.\n‘DNA replication initiates at around 4–5 hours, consistent with the expected expression timing of T7\nreplication proteins’\nWe thank the Reviewer for pointing out the inaccuracy of this statement. The statement that DNA replication\ninitiates around 4–5 hours was based directly on our experimental observations from original Figure 4a,\nrather than on well-established timing from wild-type T7 or previous cell-free expression studies. However,\nin the light of the new experiments and re-evaluation of the infection kinetics we amended the relevant\nparagraph (Lines 239 - 262; not pasted here for brevity)\nThe authors compare bulk versus SC expression, but do so at a single time point. Can the authors expand\nupon this to perform kinetic experiments for both, to ensure they are comparing apples to apples.\nWe thank the author for pointing out this issue. While we agree that kinetic information between bulk and\nencapsulated T7 assembly is important, for these experiments, endpoints are relevant since they estimate\nfinal yields between two different systems.\nWe nonetheless performed additional experiments - Fig. 4B and S31 for a kinetic comparison between\nbulk (blue bars) and compartmentalized (gray bars) CFE at 30C for T7 genome concentration of 0.1 nM\n(Supplementary Fig. S31). We observe no major difference in packaging kinetics between bulk and\nencapsulated.\nFig. S31:\nWe thank the Reviewer for this suggestion. The raw data corresponding to the fitted curves in Figure 4 are\nprovided in Supplementary Figure S35. In the light of the new experiments, we nonetheless decided to\nreplace fig 4A by the new figures 4A and 4B (see below).\nFor Fig. 4F, we improved the analysis. In the previous version of 4F liposome leak was binarized in 0 (no\nleak) or 1 (leak) so individual data points (0 and 1) were not shown. This is now improved in the new Fig\n4H where the leak ratio was quantified in >50 liposomes per condition.\nWe thank the Reviewer for this pointing this out. The calculation of PFU/nL is indeed important for interpreting\nour results and comparing phage production efficiency between bulk and liposome-based systems. For\nboth bulk and in liposomes, the PFU were recorded by performing plaque assays on serial dilutions. For\nbulk cell-free reactions, PFU/nL was determined from a 10 µL reactions. For liposome experiments,\nindividual liposome radii were measured, and assuming spherical geometry, the volume of each liposome\nwas estimated. The total encapsulated cell-free volume was then calculated by summing the volumes of all\nliposomes in the well. PFU counts were divided by this total volume, yielding PFU per nanoliter. We chose\nthe nanoliter unit as it better reflects the small total volume of encapsulated cell-free reaction. This\ncalculation is described in the Methods section (Lines 521-526):\nDetermination of PFU per nL (PFU/nL)\nFor bulk cell-free reactions, PFU/nL titers were determined from 10 µL reactions. For liposome\nexperiments, individual liposomes’ radii were measured under the microscope, and assuming\nspherical geometry, the volume of each liposome was calculated. The total encapsulated CFE\nreaction volume was then calculated by summing the volumes of all liposomes in the well. PFU\ncounts were divided by this total volume, yielding PFU per nanoliter.,\nand now clearly referenced in both the main text and figure legends for clarity.\nFor the DNA concentration studies encapsulating T7 genomes (wt and mutants), what is the % of liposomes\nexpressing mCherry as a function of DNA concentration, and how does this compare to T7 transduction in\nearlier studies. Could this give insight into what the bottleneck is?\nIndeed, it is challenging to infer the number of genomes entering a single liposome based on mCherry\nexpression due to non-linearity, variance in concentration due to heterogeneous liposome sizes, efficiency\nof encapsulation, etc.\nThe new experiments and data we now provide shed light on the potential bottlenecks. Both DNA entry and\nphage packaging are less efficient, and their kinetics is significantly slower. See amended paragraph Lines\n239-262.\n.\nWhat is the red vertical line in Figure 4a?\nThis vertical used to correspond to the inflection point of the sigmoidal fit of mCherry expression inside\nliposomes. We now removed this plot considering the new data provided, this plot is now Supplementary\nFig. S35.\nWhy do authors think wt T7 was more efficient plus LPS.\nWe indeed observe more PFU per liposome in liposome +RdLPS (~0.5) than in liposome -RdLPS (0.1-0.2).\nWhile the same CFE is encapsulated, we think liposome composition might explain this difference. Lipid\ncomposition is known to affect liposome polydispersity and encapsulation efficienc. In this case, we\ngenerally observe than liposomes +RdLPS tend to be bigger and less leaky than -RdLPS.\nRegarding LPS inhibition of mutant T7. Was this caused by DNA release or irreversible phage binding?\nWhat if these experiments were performed at 37, 42, or even 50oC. The authors could quantify free DNA\nversus encapsulated DNA by adding DNase and comparing qPCR results, exc.\nAlthough that could seem surprising, free LPS induces T7 DNA release in vitro. This is well studied and\ndocumented in this study (1). T7 is not the only case. Other phages (e.g. lambda (2), T5 (3)) in presence\nof their receptor were reported to eject their genome in solution.\n(1) González-García VA, Pulido-Cid M, Garcia-Doval C, Bocanegra R, van Raaij MJ, Martín-Benito J,\nCuervo A, Carrascosa JL. Conformational changes leading to T7 DNA delivery upon interaction with the\nbacterial receptor. J Biol Chem. 2015 Apr 17;290(16):10038-44. doi: 10.1074/jbc.M114.614222. Epub 2015\nFeb 19. PMID: 25697363; PMCID: PMC4400320.\n(2) Van Valen D, Wu D, Chen YJ, Tuson H, Wiggins P, Phillips R. A single-molecule Hershey-Chase\nexperiment. Curr Biol. 2012 Jul 24;22(14):1339-43. doi: 10.1016/j.cub.2012.05.023. Epub 2012 Jun 21.\nPMID: 22727695; PMCID: PMC3462812.\n(3) Mangenot S, Hochrein M, Rädler J, Letellier L. Real-time imaging of DNA ejection from single phage\nparticles. Curr Biol. 2005 Mar 8;15(5):430-5. doi: 10.1016/j.cub.2004.12.080. PMID: 15753037.\nCould dissipation be due to photobleaching:\n‘Further, this approach enabled visualizing genome concatemer packaging 260 maturation into fluorescent\nphage capsids. Initially, green fluorescence clusters appeared inside 261 the liposomes within the\ncharacteristic time of DNA ejection and replication (fig. 4A, movie S5), 262 indicating fluorescent capsids\nlocalized near DNA concatemers (fig. 4E, fig. S31). Over time, the 263 clusters dispersed into smaller\nfluorescence foci, suggesting concatemer processing into progeny 264 phage particles.’\nWe argue that dissipation is not due to photobleaching. For example, we plot the average intensity from\ntwo liposomes infected by T7-Split-S* over time (1 slice = 15 minutes) and show no significant decrease\nover time of the average intensity after intensity reached plateau. This is generally true for all our time-lapse\nmicroscopy where low intensity and minimal exposure were controlled to avoid bleaching.\nLiposome 1: Liposome 2:\nI believe the title and some of the claims are overreaching because lysis has to be induced.\nWe respectfully disagree with the reviewer on this point. In a way, T7 also induces lysis as the natural E.\ncoli physical lysis is achieved by an osmotic shock and not by phage proteins. We reproduce this osmotic\nshock in our work. Our results show that the cycle is complete: phage binding, DNA entry, genome\nreplication, packaging of new phages, and lysis by osmotic shock.\nIndeed, also in the natural system, the mechanism of phage-induced lysis requires a non-isotonic system.\nIn bacteria, this mechanical resistance is achieved by the cell wall - while phage lysis proteins only induce\nthe burst by degrading the cell wall - turning the bacterial cell into a spheroplast (see lysis mechanism in\nthe next answer). Our SCs, lacking a cell-wall equivalent, are hence analogous to a spheroplast by\nconstruction rendering gene encoded lysis ineffective. While we agree that incorporating a synthetic cell\nwall to provide structural integrity and an inner membrane (devoid of LPS) would be an important step\ntoward enabling gene-encoded phage release, but one that is beyond the scope of this study.\nI think ‘transduction’ instead of infection is more accurate:\nThis study demonstrates that LPS 323 alone is a sufficient membrane component for T7 genome\ninternalization into SCs, providing 324 insights into the minimal requirements for phage infection.\nWe respectfully disagree with the reviewer, since we fully demonstrated all the steps of the cycle:\ndemonstrate not only DNA entry, but its replication, packaging into new phages and lysis (as explained\nabove). We also further detailed in the revised manuscript the limitations of the cycle, in terms of the\nefficiency of the different steps and point to further improvements that are needed to achieve a system\ncapable of multiple cycles and phage evolution (Discussion, Lines 380-389):\nAchieving a self-amplifying SC phage cycle with a greater burst size would require optimization\nof several critical steps, including DNA translocation efficacy, genome replication, minimization\nof phage rebinding and engineering of the phage genome to express an enzyme capable of\nlysing the LPS membrane of the SCs. With such improvements, this system could be expanded\nto achieve multiple infection cycles and evolutionary experiments. While external intervention\nfor phage release may limit selection on the lysis cassette, potentially leading to its deletion,\nsuch a cycle would offer a unique advantage by allowing experimental control over phage\npopulation dynamics, including the suppression of fast replicating 'cheaters.' This setup could\nenable targeted selection for traits at the membrane such as tail fiber mutations, or in the\ncytoplasm such as defense systems, facilitating directed evolution of host specificity.\nPlease cite the work: ‘Unlike previous liposome studies with T5, which lacked cytoplasmic CFE’\nThank you. The citation was added:\nLambert, O., Plançon, L., Rigaud, J. L. & Letellier, L. Protein-mediated DNA transfer into liposomes. Mol\nMicrobiol 30, 761–765 (1998)\nI am confused by ‘low multiplicity of infection’. Doesn’t the system require high MOI:\n‘The low multiplicity of infection 343 (MOI) constrains the number of productive infections, while DNA\nreplication in CFE achieves 344 2–3-fold amplification compared to the 200–300-fold observed in bacterial\nhosts due to finite 345 resources in CFE.’\nWe thank the Reviewer for noticing this mistake. We corrected this sentence as follows (Lines 400-403):\n“The large multiplicity of infection (MOI around 100) constrains the number of productive\ninfections, while DNA replication in CFE achieves 1–2-fold amplification compared to the 200–\n300-fold observed in bacterial hosts due to finite resources in CFE”\nThis manuscript describes the use of cell free machinery in a membrane encapsulation to study phage\nreplication. The presentation is excellent, with each step of the phage replication process carefully validated\nand analyzed. The experimental design is rigorous and described in a very detailed way, offering high\nnovelty and significant potential for advancing both phage biology and phage engineering. The schematics\nare really helpful for understanding the experimental setup and interpreting results, although sometimes the\nexplanations given in the figure legends are clearer than what is presented in the text. Overall, this work\nis important given the dearth of knowledge surrounding phage and their promise as novel therapeutics.\nSome minor clarifications, detailed below, would enhance the overall quality and significance of the work.\nWe thank the Reviewer for their positive assessment.\n78-80: “to prevent unintended genome ejection” Was this observed or only hypothesized?\nWe thank the Reviewer for this question. The statement is based on a design hypothesis rather than a\ndirect observation. Since LPS serves as the receptor for T7, there is a logical concern that newly produced\nphages inside the synthetic cells could bind internal LPS molecules if presented symmetrically on both\nsides of the membrane or preferentially oriented towards the CFE inside the liposome. This interaction\ncould potentially trigger premature genome ejection within the liposome, inhibiting newly assembled virions.\nWhile we have not directly observed such events, the preparation of liposomes was designed to prevent\nthis possibility. It’s worth noting that in native E. coli, the inner membrane does not contain LPS, further\nsupporting our rationale for minimizing internal LPS exposure in this system.\n80: “RdLPS” Define this abbreviation; where possible, use fewer abbreviations throughout to enhance the\nflow and readability.\n81: \"unlike wild-type T7 phage (T7 WT)\" Please clarify which type of LPS the T7 WT can interact with.\nWe addressed comments as follows (Lines 80-84):\n“Accordingly, we opted for RdLPS (LPS of the Rd chemotype containing only the inner core; Fig.\n1A), a short rough LPS type (Fig. S1). To enable interaction with this minimal receptor, we used a\ncognate T7 phage tail fiber mutant that can bind RdLPS in vitro, unlike wild-type T7 (T7 WT),\nwhich binds only to RaLPS (containing both inner and outer core; Fig. S1).”\n95-96: \"a chimeric protein (GFP-TF*) consisting of superfolder GFP fused to the N-terminal of the tip of the\nT7 RdLPS-specific tail fiber mutant (TF*)\" The description is confusing. Does GFP-TF* only contain the tip\nof the phage tail fiber or the whole mutant?\nWe clarified this point as follows (Lines 96-99):\n\"Incorporation of RdLPS into the outer leaflet of the lipid membrane and its expected function were\nassayed using a chimeric protein (GFP-TF*) consisting of superfolder GFP fused to the N-terminus\nof the C-terminal 'tip' domain of the T7 tail fiber mutant (TF*), which includes the S541R mutation\nresponsible for RdLPS recognition (Fig. S4).\"\n158: \"LPS were homogeneously distributed across liposomes, not all liposomes were infected \" Including\nthe explanation in figure S8B – \"Only a fraction of RdLPS SCs binding GFP-TF* appear red, due to SCs\nleak, disruption or inactive SCs\" – would greatly clarify things here.\nWe amended this sentence (Lines 161-163):\n“The addition of GFP-TF* to the infected RdLPS SCs revealed that while RdLPS were\nhomogeneously distributed across liposomes, not all liposomes were infected due to SCs leak,\ndisruption or inactive CFE (fig. 3C)”\n170: “intercalating fluorescent dye” Please specify.\nTo address this point, we made the following change (Lines 178-180):\n“We visualized genome entry into the SCs using SYBR Gold DNA intercalating fluorescent dye\nthat does not inhibit CFE (Fig. 3H, Supplementary Fig. S21-24, movie S3)”\n.174: \"these clusters dissipated as mCherry fluorescence increased\" The mCherry fluorescence is not\nconsistent with the previous description of \"Upon the addition of T7-S* ….\" since the phage T7-S* shouldn't\nexpress mCherry during replication. Please clarify.\nour statement as follows (Line 180-181):\n“Upon the addition of T7-mC-S* (MOI ≈100) in the outer solution, an increase in fluorescence\nintensity inside the SCs was observed along with the formation of fluorescent clusters (fig. 3I, fig.\nS24), indicating genome entry.”\n210: Consider describing TXTL for clarity.\nWe decided to use CFE (cell-free gene expression) rather than TXTL for overall clarity. The 6 TXTL\ninstances were changed to CFE.\n229-230: Please specify that the kinetics of three probes are generated independently instead of in\ncombination.\nFor clarity, this paragraph was entirely revised and rewritten, as shown in the revised manuscript (Lines\n239-262).\n242-243: \"At the lowest DNA concentration (10 pM), phage synthesis was more prevalent inside the SCs\nthan in batch mode, likely due to favorable colocalization of gene expression machinery under confinement\nas compared to batch reactions\" Alternatively, the reason might be the colocalization of the phage\ncomponents for assembly instead of the colocalization of gene expression machinery.\nWe thank the Reviewer for pointing this out. This comment is indeed very relevant. The sentence now reads\nas (Line 267):\n“At the lowest DNA concentration (10 pM), phage synthesis was more prevalent inside the SCs\nthan in batch mode, likely due to favorable colocalization of gene expression machinery and\nphage components under confinement as compared to batch reactions.”\n246-248: \"Next, to assess the effect of RdLPS incorporation on phage titer, we encapsulated T7-WT or T7-\nmC-S* genomes (10 pM) inside SCs with or without RdLPS into the membrane\" Why not use T7-\nWT and T7-S* for comparison?\nWe thank the Reviewer for this comment. We chose to use T7-mC-S* rather than T7-S* in this experiment\nbecause the mCherry reporter provides a useful internal control for gene expression, allowing us to visually\nconfirm successful infection and expression in individual synthetic cells via red fluorescence. While we\nacknowledge that T7-mC-S* is not strictly identical to T7-S* due to the added genetic payload, the focus of\nthis experiment was not on absolute PFU values but on the relative effect of RdLPS incorporation.\nSpecifically, we observed no significant difference in the ratio of PFUs produced with or without RdLPS in\nthe membrane, supporting the conclusion that phages rebind to available LPS after release.\n288: Why are individual data points not shown in A and F?\nFig.4A has been replaced. For Fig. 4F, we improved the analysis. In the previous version of 4F liposome\nleak was binarized in 0 (no leak) or 1 (leak) so individual data points (0 and 1) were not shown. This is now\nimproved in the new Fig 4H where the leak ratio was quantified in >50 liposomes per condition.\n26\n298: Why was there a discrepancy in the number of replicates (3 vs 4)?\nWe did not perform 3 replicates in batch mode and 4 replicates for a rational reason, only experimental\nconvenience.\n323-324: \"This study demonstrates that LPS alone is a sufficient membrane component for T7 genome\ninternalization into SCs\" This claim might not be incorrect, but to be more rigorous, it would be better to use\n\"RdLPS\" and \"T7-S*\" specifically for this study.\nWe thank the Reviewer for this suggestion, with which we agree. The sentence now reads as (Line 364):\n\"This study demonstrates that RdLPS alone is a sufficient membrane component for T7-S*\ngenome internalization into this SC system\"\n344-345: \"DNA replication in CFE achieves 2–3-fold amplification\". Is this claim from another publication?\nIt would be informative to show the DNA replication data in this study.\nThis number is based on a previous work done by our group on DNA replication (Shin, J., Jardine, P., &\nNoireaux, V. (2012). Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-\nfree reaction. ACS synthetic biology, 1(9), 408-413.). We now quantified the value in our specific setting to\nbe around 1.5x factor.\nFor 0.1 nM of T7 genome encapsulated in SCs at 30C, we recover, after 5 hour of incubation, 9±3x10^4\nPFU/nL, as compared to a theoretical value of 6x10^4 PFU/nL expected if all genomes were fully packaged\ninto viable phages. We hence obtain slightly higher titer values than expected suggesting a DNA replication\nfactor of about 1.5 in SCs at 30C. This gives us a lower limit of the average replication efficiency, as phage\ngenome purification does not result in perfect integral genomes and packaged non-viable packaged phages\nwill not propagate to be counted as PFUs with the agar overlay method possibly underestimating about 2\ntimes the replication factor.\n348: “substantial challenges” Please elaborate on this.\nWe added upstream in the discussion a separate paragraph on the limitations and future challenges of the\nsystem (Lines 405-412). We thus removed the first part of the addressed sentence, it now reads: This work\noffers ….’.\n361-363: “Biocontainment” is not mentioned elsewhere in the manuscript. It seems a non sequitur to\nintroduce it here.\nWe hold a different view which we believe is defendable Here, we highlight another potential future aspect\nof the work presented in this manuscript, contributing a design principle for SCs (integrating phage receptor)\nto be infectable by phages and thus contained.\nThis is thus important in the context of rising safety concerns regarding synthetic cells. We slightly modified\nthese lines to make the point clearer (Lines 418-419):\n27\n‘Biocontainment solutions must be anticipated and imagined, considering that bottom-up SCs\nare currently not designed to be infectable by phages. In that respect, the cell-free phage\ninfection mechanism devised in this work delivers fundamental information for developing one\ntype of biocontainment solution to control SC systems’.\n435-436: \"custom-made programs on Python\" Please ensure the scripts are available.\nThe scripts for plots and analysis will be uploaded on GitHub with final submission.\n453: What is the ratio between reaction buffer and cell extract? Or is the reaction buffer used for the cell\nlysis? Please clarify.\nThe sentence now reads as: (Line 510)\n“The volume of the CFE reaction consisted of the 33% cell extract, 67% of the other components\n(energy mix, amino acids).”\n456-457: \"Amino acids were added at concentrations between 1.5 mM and 3 mM for each of the 20 amino\nacids.\" Please add a supplemental table for this.\nThis is now added in Supplementary Data 1.\n500: \"Liposomes were randomly selected\" How was \"randomly\" achieved?\nLiposomes were picked by a lab member unbiased by any prior knowledge related to the system studied,\nto curate the data and improve the analysis. The lab member was asked to pick liposomes that are entirely\ncontained within the imaging frame and that have a well-defined outline that does not overlap with other\nliposomes. When possible, liposomes were selected on a channel containing the volume marker and not\nthe signal of interest.\n541: \"average of the last ten images colorized green\" Where does the green come from?\nGreen corresponds to an artificial coloring of the same red fluorescent dye but at the end of the experiment\n(average of the last 50 minutes from 2:10 - 3:00). We added ‘artificially’ (Line 621).\n542-543: \"red liposomes (leaked liposomes) and orange liposomes (no leak)\" Why does the red refer to\n\"leaked liposomes\" and the orange to \"no leak\"?\n“Red liposomes” = corresponds to liposomes that contain the dye in the beginning (colorized red) and no\ndye at the end (no signal); so the superposition of the two image averages is red. the liposomes leaked\nupon holin expression.\n“orange liposomes” = corresponds to liposomes that contain the dye in the beginning (colorized in red) and\nat the end (colorized in green); so the superposition of the two image averages is orange. the liposome did\nnot leak upon holin expression.\nThe method description was improved to reflect this. Red and green signal were averaged in the liposomes\nand the Leak ratio=1-(I_f-BG_f)/(I_i-BGi ) where I_i and I_f are the average fluorescence intensities inside\nthe liposomes measured respectively right after creation and at the end of the incubation, while BG_i and\nBG_f and the corresponding background fluorescence intensities.\nThe corresponding plot and method section was updated.\nFigure 3G: Which MOI was used for this scatter plot? If the MOI were changed, would the result be different?\nWe thank the Reviewer for this question. The scatter plot in Figure 3G was generated using an MOI of\napproximately 300, which we identified as optimal for efficient liposome infection in our system. We should\nexpect the observed effect to be minimized at high MOI, where most or all liposomes, regardless of size,\nreceive at least one phage genome, thereby saturating the population. Conversely, at lower MOI, infection\nwould be more stochastic, and smaller liposomes (with a higher surface-to-volume ratio) would be more\nlikely to show signs of infection, such as mCherry expression, due to increased likelihood of successful\nDNA injection per unit volume.\nTo further study this effect, we performed a more complete analysis of mCherry concentration in SCs as a\nfunction of radius across MOI values (3000, 300, 30 and 3) (Supplementary Fig. S20). We observe the\nsame trend, with smaller MOI favoring smaller liposomes while high MOI saturates the liposomes with\nphage genomes.\nto RdLPS-. Any explanation?\nThis was changed. This could be due to the liposome membrane differences inducing changes in liposome\npolydispersity and encapsulation efficiency. We noticed that liposomes with LPS tend to be bigger.\nFigure S4C: \"membrane without LPS\" Change LPS to RdLPS.\nDone.\nFigure S13C (right): Any explanation for the huge variation in mCherry signal?\nThis signal corresponds to mCherry fluorescence not concentration so this signal is liposome size\ndependent. Hence bigger liposomes have bigger signals than smaller ones.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nReviewer #1 (Remarks to the Author):\nNA.\nReviewer #2 (Remarks to the Author):\nWe thank the reviewer for his/her comments and appreciation of the work.\nReviewer #3 (Remarks to the Author):\nThe authors have provided a greatly revised manuscript with careful responses that generally\naddress all concerns. A few minor clarifications:\n227: It should be T7-mC-S* instead of T7-S*\nThank you for catching this, this was corrected.\n272-273: Maybe not necessary, but maybe clarify that the RdLPS is facing towards outer leaflet\n?\nThe sentence now reads as: ‘Next, to assess the effect of RdLPS incorporation on phage titer, we encapsulated T7-WT or T7-mC-S* genomes (10 pM) inside SCs with or without RdLPS into the membrane (the RdLPS facing toward the outer solution).’\n625: Clarify \"GFP\"\nThank you for catching this. This was changed to ‘red fluorescent dextran dye’.\nRegarding the comment about LPS inhibition of mutant T7, irreversible phage binding could still\noccur, which might account for some of the inefficiencies.\nWe agree with this point, the sentence (line 202-204) now reads as: “However, phage titers are\nreduced for RdLPS-susceptible phages, likely due to inactivation by irreversible phage binding to\nRdLPS.”\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nNA.\n1"}]

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