AbstractReplication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging.

复制、遗传和进化是生命的特征。我们和其他人假设，实验室中合成生命系统的重建将取决于能够经历达尔文进化的遗传自我复制因子的发展。尽管基于DNA的生命占主导地位，但进化中的DNA自我复制子的体外重建仍然具有挑战性。

We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template – encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage – expressed in the ‘protein synthesis using recombinant elements’ (PURE) system.

我们在此在脂质体隔室中模拟生命传播信息和进化的原理。使用支持间歇性或半连续进化的两种不同实验配置（即有或没有DNA提取，PCR和重新包封），我们证明了线性DNA模板的可持续复制-编码来自Phi29噬菌体的DNA聚合酶和末端蛋白-在“使用重组元件的蛋白质合成”（PURE）系统中表达。

The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations.

自我复制因子可以在脂质体中的多轮复制偶联转录-翻译反应中存活，并且仅在十轮进化中积累赋予选择优势的突变。将来自下一代测序的数据与一些富集突变的反向工程相结合，揭示了引入突变的非平凡和上下文依赖性影响。

The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system..

目前的结果为在进化的合成细胞中建立遗传复杂性以及在最小无细胞系统中研究进化过程奠定了基础。。

IntroductionA fundamental goal of modern synthetic biology is the construction of synthetic systems with life-like properties. It is also likely that the learning-by-doing approach involved in the construction of a synthetic cell will spearhead advances in biomedicine, biotechnology, and fundamental biology1,2,3,4,5,6.

引言现代合成生物学的一个基本目标是构建具有生命特性的合成系统。参与合成细胞构建的边干边学方法也可能引领生物医学，生物技术和基础生物学的进步1,2,3,4,5,6。

In this quest, a number of life’s features have been reconstituted in a cell-free environment7,8,9,10,11,12,13, although a functionally integrated, autonomous synthetic cell seems still out of reach.One remarkable feature of extant living forms is evolution, i.e., the ability to diversify and gradually adapt to changing environments.

在这项研究中，许多生命特征已经在无细胞环境中重建7,8,9,10,11,12,13，尽管功能整合的自主合成细胞似乎仍然遥不可及。现存生物形式的一个显着特征是进化，即多样化并逐渐适应不断变化的环境的能力。

This ability is responsible for terrestrial Life’s extraordinary diversity and robustness, allowing it to colonize extremely diverse niches and survive multiple geological calamities in the past 3.5–3.8 billion years14,15. Importantly, it is thought that evolution, in an abiotic, molecular version also has allowed life’s emergence in the first place16,17,18,19,20.

。重要的是，人们认为，在非生物的分子版本中，进化首先也允许生命的出现16,17,18,19,20。

We therefore asked whether in vitro evolution could be used as a tool in our efforts to build a synthetic cell and better understand living processes.A key prerequisite for evolution is heredity. In contemporary life form, the dominant molecular mechanism supporting heredity is DNA replication21. In vitro reconstitution of replication thus represents a major step in crafting synthetic living systems from the ground up6.

因此，我们询问体外进化是否可以作为我们构建合成细胞和更好地理解生命过程的工具。进化的一个关键先决条件是遗传。在当代生命形式中，支持遗传的主要分子机制是DNA复制21。因此，复制的体外重建代表了从底层构建合成生命系统的重要一步6。

However, early molecular replicators could in principle have been based on molecules other than DNA22,23. A variety of self-replicating non-DNA systems have been created in the laboratory. For example, non-enzymatic self-replication based on autocatalytic template production24,25, cross-catalysing RNA replicators26, self-replicating peptides27,28,29,.

然而，早期的分子复制子原则上可能基于DNA22,23以外的分子。实验室中已经创建了各种自我复制的非DNA系统。例如，基于自催化模板产生的非酶促自我复制24,25，交叉催化RNA复制子26，自我复制肽27,28,29，。

Next, we investigated whether it is possible to minimize the researcher intervention in the in vitro evolution process via a more streamlined evolution protocol. In such a system, amplified DNA from a fraction of liposomes would be recursively passed on to an excess of fresh liposomes via vesicle fusion and fission.

接下来，我们调查了是否有可能通过更简化的进化协议最大限度地减少研究人员对体外进化过程的干预。在这样的系统中，来自一部分脂质体的扩增DNA将通过囊泡融合和裂变递归传递给过量的新鲜脂质体。

This scheme would obviate out-of-liposome PCR amplification and controlled re-encapsulation of DNA (Fig. 3a). Fresh feeding vesicles supply additional lipids, PURE components, and additives for DNA replication. Both fusion and fission events are promoted by freeze-thaw (F/T) cycles, during which pooling and stochastic redistribution of the DNA content are expected.

该方案将避免脂质体PCR扩增和DNA的受控重新包封（图3a）。新鲜喂养囊泡为DNA复制提供额外的脂质，纯成分和添加剂。冻融（F/T）循环促进了融合和裂变事件，在此期间，预计DNA含量会汇集和随机重新分布。

A similar protocol was used by Tsuji et al. for the replication of RNA over multiple rounds of liposome cultivation60. We called this evolution scheme “semi-continuous”, as it is a step towards continuous in vitro evolution, where a synthetic cell can pass on its genetic information to the next generation without researcher intervention5.

Tsuji等人使用类似的方案在多轮脂质体培养中复制RNA 60。我们将这种进化方案称为“半连续”，因为它是迈向连续体外进化的一步，在这种进化中，合成细胞可以将其遗传信息传递给下一代，而无需研究人员的干预5。

Noteworthily, the fact that the output of the selection process is exactly the same molecule as the input is a critical feature to enable continuous evolution.Fig. 3: Semi-continuous evolution of mod-ori-p2p3.a Schematic illustration of the experimental set-up for a semi-continuous evolution approach in liposomes.

值得注意的是，选择过程的输出与输入完全相同的分子这一事实是实现连续进化的关键特征。图3:mod-ori-p2p3的半连续进化。脂质体中半连续进化方法的实验装置的示意图。

Step (i) in-liposome IVTTR (ii) 100-fold dilution of old vesicle suspension in a suspension of fresh vesicles, (iii) liposome fusion-fission promoted by cycles of F/T. b Trajectories of mod-ori-p2p3 concentrations in liposomes in the semi-continuous evolutionary campaign, Cont-WT, as measured by qPCR.

步骤（i）在脂质体IVTTR中（ii）将新鲜囊泡悬浮液中的旧囊泡悬浮液稀释100倍，（iii）由F/T循环促进的脂质体融合裂变。通过qPCR测量，脂质体中mod-ori-p2p3浓度的轨迹在半连续进化活动中，Cont-WT。

Each IVTTR was incubated for 16 h and liposomes were diluted 100 times with feeding vesicles. The target region for qPCR quantification ( ~ 200 bp) belong.

将每个IVTTR孵育16小时，并用喂食囊泡将脂质体稀释100倍。qPCR定量的目标区域（〜200 bp）属于。

Unlabelled nucleotides were purchased from GE Healthcare. The [γ-32P]ATP (3,000 Ci/mmol) and [α-32P]dATP (3000 Ci/mmol) were supplied by PerkinElmer. Oligonucleotides sp1 (5ʹ-GATCACAGTGAGTAC), sp1c + 6 (5ʹ-TCTATTGTACTCACTGTGATC), and M13 Universal Primer (5ʹ-GTAAAACGACGGCCAGT) were purchased from Sigma-Aldrich.

未标记的核苷酸购自GE Healthcare。[γ-32P]ATP（3000 Ci/mmol）和[α-32P]dATP（3000 Ci/mmol）由PerkinElmer提供。寡核苷酸sp1（5'-GATCACAGTGAGTAC），sp1c+ 6（5'-TCTATTGTACTCACTGTGATC）和M13通用引物（5'-GTAAAACGACGGCCAGT）购自Sigma-Aldrich。

T4 polynucleotide kinase (T4PNK) was purchased from New England Biolabs. Oligonucleotide sp1 was 5ʹ-labelled with 32P using [γ-32P]ATP (10 μCi) and T4PNK and further hybridised to oligonucleotide sp1c + 6 (1:2 ratio) to get the primer/template substrate sp1/sp1c + 6 for the Exonuclease/Polymerisation balance assays (see below).

T4多核苷酸激酶（T4PNK）购自New England Biolabs。使用[γ-32P]ATP（10μCi）和T4PNK用32P标记寡核苷酸sp1，并进一步与寡核苷酸sp1c+6（1:2比例）杂交，得到引物/模板底物sp1/sp1c+6用于核酸外切酶/聚合平衡测定（见下文）。

Oligonucleotides were annealed in the presence of 50 mM Tris-HCl (pH 7.5) and 0.2 M NaCl, heating to 90 °C for 10 min before slowly cooling to room temperature overnight. M13mp18 (+) strand ssDNA (Sigma-Aldrich) was hybridized to the universal primer as described above, and the resulting molecule was used as a primer/template complex to analyse processive DNA polymerisation coupled to strand displacement by the wild-type and variants of Φ29 DNAP.

寡核苷酸在50mM Tris-HCl（pH 7.5）和0.2M NaCl存在下退火，加热至90℃10分钟，然后缓慢冷却至室温过夜。如上所述，将M13mp18（+）链ssDNA（Sigma-Aldrich）与通用引物杂交，并将所得分子用作引物/模板复合物，以分析与野生型和Φ29 DNAP变体的链置换偶联的进行性DNA聚合。

Terminal protein-Φ29 DNAP complex (TP-DNA) was obtained as described in ref. 86.Primed M13 DNA replication assayThe incubation mixture contained, in 25 μL, 50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 4% (v/v) glycerol, 0.1 mg/mL of BSA, 40 μM dNTPs and [α-32P]dATP (1 μCi), 4.2 nM of primed M13mp18 ssDNA, and 60 nM of either the wild-type or the indicated mutant Φ29 DNA polymerase.

如参考文献86所述获得末端蛋白-Φ29 DNAP复合物（TP-DNA）。引发的M13 DNA复制测定孵育混合物含有25μL，50mM Tris-HCl，pH 7.5，10mM MgCl2，1mM DTT，4%（v/v）甘油，0.1mg/mL BSA，40m dNTPs和[α-32P]dATP（1μCi），4.2nM引发的M13mp18 SSP DNA和60 nM的野生型或指定的突变Φ29 DNA聚合酶。

After incubation at 30 °C for the indicated times, the reactions were stopped by adding 10 mM EDTA-0.1% SDS and the samples were filtered through Sephadex G-50 spin columns. For size analyses of the synthesised DNA, the labelled DNA was denatured by treatment with 0.7 M NaOH and subjected to electrophoresis in alkalin.

在30℃温育指定时间后，通过加入10mM EDTA-0.1%SDS终止反应，并通过Sephadex G-50旋转柱过滤样品。为了对合成的DNA进行大小分析，通过用0.7M NaOH处理使标记的DNA变性，并在碱性中进行电泳。

Data availability

数据可用性

Data are available in the main manuscript, Supporting Information, and Supplementary Data 1. Protein mass spectrometry data are available on Panorama Public under ProteomeXchange ID PXD054024 and accession URL: https://panoramaweb.org/qxuWjQ.url. Raw NGS sequencing data have been uploaded to ENA (European Nucleotide Archive) under project_ID PRJEB75735.

。蛋白质质谱数据可在Panorama Public上以ProteomeXchange ID PXD054024和登录URL获得：https://panoramaweb.org/qxuWjQ.url.原始NGS测序数据已在project\u ID PRJEB75735下上传到ENA（欧洲核苷酸档案馆）。

The previously published protein structure used in this work is available from the Protein Data Bank under ID 2EX3. Raw data in multiple labelled files (Excel and GraphPad) are available within a zipped folder named ‘Source Data’. Source data are provided with this paper..

这项工作中使用的先前发布的蛋白质结构可从ID 2EX3的蛋白质数据库中获得。多个标签文件（Excel和GraphPad）中的原始数据可在名为“源数据”的压缩文件夹中找到。。。

Code availability

代码可用性

Galaxy workflow for mapping and quantifying mutation frequencies is available on the GitHub repository (https://github.com/DanelonLab/Illumina-NGS-Mutation-Mapping) and zenodo, with https://doi.org/10.5281/zenodo.13757341.

GitHub存储库中提供了用于映射和量化突变频率的Galaxy工作流程(https://github.com/DanelonLab/Illumina-NGS-Mutation-Mapping)和zenodohttps://doi.org/10.5281/zenodo.13757341.

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Download referencesAcknowledgementsWe would like to thank Ilja Westerlaken for single clone preparation and Sanger sequencing analysis, Elisa Godino for assisting with the characterization of some DNA variants, Anna de Jong for the flow cytometry experiments, and Laura Sierra Heras for performing a replicate of Bulk-WT.

下载参考文献致谢我们要感谢Ilja Westerlaken进行单克隆制备和Sanger测序分析，感谢Elisa Godino协助表征某些DNA变体，感谢Anna de Jong进行流式细胞术实验，感谢Laura Sierra Heras进行Bulk-WT的复制。

The research was funded by the NWO Gravitation programs “BaSyC – Building a synthetic cell” (024.003.019) and “NanoFront – Frontiers of Nanoscience”. CD acknowledges funding from ANR (ANR-22-CPJ2-0091-01). ZA acknowledges funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no.

这项研究由NWO引力项目“BaSyC-构建合成细胞”（024.003.019）和“纳米前沿-纳米科学前沿”资助。CD承认ANR（ANR-22-CPJ2-0091-01）的资助。ZA感谢欧盟地平线2020研究与创新计划根据MarieSkłodowska Curie grant agreement no.提供的资金。

707404. ARS acknowledges funding from a NanoFront-Casimir Research School PhD grant. MdV acknowledges funding by MCIN/AEI/10.13039/501100011033 (Grant PID2020-115978GB-I00). We specifically acknowledge the contributions of author Andreea R. Stan, who sadly passed away and did not see the final version of the paper.Author informationAuthor notesThese authors contributed equally: Zhanar Abil, Ana María Restrepo Sierra.Deceased: Andreea R.

ARS感谢NanoFront Casimir研究院博士学位授予的资金。MdV承认MCIN/AEI/10.13039/501100011033（Grant PID2020-115978GB-I00）的资助。我们特别感谢作者AndreeaR.Stan的贡献，他不幸去世，没有看到论文的最终版本。作者信息作者注意到这些作者做出了同样的贡献：Zhanar Abil，Ana María Restrepo Sierra。死者：Andreea R。

Stan.Authors and AffiliationsDepartment of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, NetherlandsZhanar Abil, Ana María Restrepo Sierra, Andreea R. Stan, Amélie Châne & Christophe DanelonDepartment of Biology, University of Florida, 882 Newell Dr, Gainesville, USAZhanar AbilCentro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Nicolás Cabrera, 1, Madrid, SpainAlicia del Prado & Miguel de VegaLaboratoire Gulliver, UMR7083 CNRS/ESPCI Paris-PSL Research University, 10 rue Vauquelin, Paris, FranceYannick RondelezToulouse Biotechnology Institute (TBI.

斯坦。生物纳米科学的作者和附属机构，卡夫利纳米科学研究所，代尔夫特理工大学，代尔夫特，荷兰萨纳尔·阿比尔，安娜·玛丽亚·雷斯特雷波·塞拉，安德里亚·R。斯坦，阿梅莉·查内和克里斯托夫·达内隆德生物系，佛罗里达大学，882纽厄尔博士，盖恩斯维尔，乌萨扎纳尔·阿比尔分子生物学中心塞韦罗·奥乔亚（马德里自治大学高级科学研究委员会），尼古拉斯·卡布雷拉，1岁，马德里，Spainalicia del Prado&Miguel de Vegalabaratoire Gulliver，UMR7083 CNRS/ESPCI Paris-PSL Research University，10 Rue Vauquelin，Paris，Franceyannick RondelezToulouse Biotechnology Institute（TBI）。

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PubMed Google ScholarContributionsCD conceived and supervised the project; ZA, AMRS, ARS, AC, and AdP designed, performed experiments, and performed data analysis; MdV designed experiments and performed data analysis; YR performed data analysis; ZA, AMRS and CD wrote the paper with input from all co-authors.Corresponding authorCorrespondence to.

PubMed谷歌学术贡献SCD构思并监督了该项目；ZA，AMRS，ARS，AC和AdP设计，进行实验并进行数据分析；MdV设计实验并进行数据分析；YR进行了数据分析；ZA，AMRS和CD在所有合著者的意见下撰写了这篇论文。对应作者对应。

Christophe Danelon.Ethics declarations

克里斯托夫·达内隆。道德宣言

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The authors declare no competing interests.

作者声明没有利益冲突。

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Nature Communications thanks Norikazu Ichihashi, Ryo Mizuuchi, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Nature Communications感谢Norikazu Ichihashi，Ryo Mizuuchi和另一位匿名审稿人对这项工作的同行评审做出的贡献。同行评审文件可用。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary informationSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummaryTransparent Peer Review fileSource dataSource DataRights and permissions.

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Reprints and permissionsAbout this articleCite this articleAbil, Z., Restrepo Sierra, A.M., Stan, A.R. et al. Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell.

转载和许可本文引用本文Abil，Z.，Restrepo Sierra，A.M.，Stan，A.R。等人。达尔文进化论在合成原细胞中自我复制DNA的进化。

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