Skip to main content
SpringerLink
Log in

Compartmentalized Cell-Free Expression Systems for Building Synthetic Cells

  • Chapter
  • First Online:
Cell-free Production

Part of the book series: Advances in Biochemical Engineering/Biotechnology ((ABE,volume 186))

Abstract

One of the grand challenges in bottom-up synthetic biology is the design and construction of synthetic cellular systems. One strategy toward this goal is the systematic reconstitution of biological processes using purified or non-living molecular components to recreate specific cellular functions such as metabolism, intercellular communication, signal transduction, and growth and division. Cell-free expression systems (CFES) are in vitro reconstitutions of the transcription and translation machinery found in cells and are a key technology for bottom-up synthetic biology. The open and simplified reaction environment of CFES has helped researchers discover fundamental concepts in the molecular biology of the cell. In recent decades, there has been a drive to encapsulate CFES reactions into cell-like compartments with the aim of building synthetic cells and multicellular systems. In this chapter, we discuss recent progress in compartmentalizing CFES to build simple and minimal models of biological processes that can help provide a better understanding of the process of self-assembly in molecularly complex systems.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 219.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Buchner E (1897) Alkoholische Gährung ohne Hefezellen. Ber Dtsch Chem Ges 30:117–124

    Article  CAS  Google Scholar 

  2. Hoagland MB, Zamecnik PC, Stephenson ML (1957) Intermediate reactions in protein biosynthesis. Biochim Biophys Acta 24:215–216

    Article  CAS  PubMed  Google Scholar 

  3. Kirsch JF, Siekevitz P, Palade GE (1960) Amino acid incorporation in vitro by ribonucleoprotein particles detached from Guinea pig liver microsomes. J Biol Chem 235:1419–1424

    Article  CAS  PubMed  Google Scholar 

  4. Fuller RS, Kaguni JM, Kornberg A (1981) Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci 78:7370–7374

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Blow JJ, Laskey RA (1986) Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of xenopus eggs. Cell 47:577–587

    Article  CAS  PubMed  Google Scholar 

  6. Nirenberg MW, Matthaei JH (1961) The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci 47:1588–1602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Matthaei JH, Nirenberg MW (1961) Characteristics and stabilization of DNAase-sensitive protein synthesis in E. coli extracts. Proc Natl Acad Sci 47:1580–1588

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nathans D, Notani G, Schwartz JH, Zinder ND (1962) Biosynthesis of the coat protein of coliphage f2 by E. coli extracts. Proc Natl Acad Sci 48:1424–1431

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. DeVries JK, Zubay G (1967) DNA-directed peptide synthesis. II. The synthesis of the alpha-fragment of the enzyme beta-galactosidase. Proc Natl Acad Sci 57:1010–1012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zubay G (1973) In vitro synthesis of protein in microbial systems. Annu Rev Genet 7:267–287

    Article  CAS  PubMed  Google Scholar 

  11. Garenne D, Thompson S, Brisson A, Khakimzhan A, Noireaux V (2021) The all- E. coli TXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform. Synth Biol 5:344–355

    Google Scholar 

  12. Des Soye BJ, Davidson SR, Weinstock MT, Gibson DG, Jewett MC (2018) Establishing a high-yielding cell-free protein synthesis platform derived from vibrio natriegens. ACS Synth Biol 7:2245–2255

    Article  CAS  PubMed  Google Scholar 

  13. Wiegand DJ, Lee HH, Ostrov N, Church GM (2019) Cell-free protein expression using the rapidly growing bacterium Vibrio natriegens. J Vis Exp. https://doi.org/10.3791/59495

  14. Kelwick R, Webb AJ, MacDonald JT, Freemont PS (2016) Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements. Metab Eng 38:370–381

    Article  CAS  PubMed  Google Scholar 

  15. Wang H, Li J, Jewett MC (2018) Development of a Pseudomonas putida cell-free protein synthesis platform for rapid screening of gene regulatory elements. Synth Biol 3:1–7

    Article  Google Scholar 

  16. Li J, Wang H, Kwon YC, Jewett MC (2017) Establishing a high yielding streptomyces-based cell-free protein synthesis system. Biotechnol Bioeng 114:1343–1353

    Article  CAS  PubMed  Google Scholar 

  17. Moore SJ, MacDonald JT, Wienecke S, Ishwarbhai A, Tsipa A, Aw R, Kylilis N, Bell DJ, McClymont DW, Jensen K et al (2018) Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria. Proc Natl Acad Sci 115:E4340–E4349

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hodgman CE, Jewett MC (2013) Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis. Biotechnol Bioeng 110:2643–2654

    Article  CAS  PubMed  Google Scholar 

  19. Takai K, Sawasaki T, Endo Y (2010) Practical cell-free protein synthesis system using purified wheat embryos. Nat Protoc 5:227–238

    Article  CAS  PubMed  Google Scholar 

  20. Ezore T, Suzuki T, Higashide S, Shintani E, Endo K, Kobayashi SI, Shikata M, Ito M, Tanimizu K, Nishimura O (2006) Cell-free protein synthesis system prepared from insect cells by freeze-thawing. Biotechnol Prog 22:1570–1577

    Article  Google Scholar 

  21. Mikami S, Kobayashi T, Masutani M, Yokoyama S, Imataka H (2008) A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expr Purif 62:190–198

    Article  CAS  PubMed  Google Scholar 

  22. Stavnezer J, Huang RCC (1971) Synthesis of a mouse immunoglobulin light chain in a rabbit reticulocyte cell-free system. Nat New Biol 230:172–176

    Article  CAS  PubMed  Google Scholar 

  23. Buntru M, Vogel S, Spiegel H, Schillberg S (2014) Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system. BMC Biotechnol 14:1–11

    Article  Google Scholar 

  24. Buntru M, Vogel S, Stoff K, Spiegel H, Schillberg S (2015) A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates. Biotechnol Bioeng 112:867–878

    Article  CAS  PubMed  Google Scholar 

  25. Eagon R (1962) Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. J Bacteriol 83:736–737

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Weinstock MT, Hesek ED, Wilson CM, Gibson DG (2016) Vibrio natriegens as a fast-growing host for molecular biology. Nat Methods 13:849–851

    Article  CAS  PubMed  Google Scholar 

  27. Hirano N, Sawasaki T, Tozawa Y, Endo Y, Takai K (2006) Tolerance for random recombination of domains in prokaryotic and eukaryotic translation systems: limited interdomain misfolding in a eukaryotic translation system. Proteins Struct Funct Bioinforma 64:343–354

    Article  CAS  Google Scholar 

  28. Tarui H, Imanishi S, Hara T (2000) A novel cell-free translation/glycosylation system prepared from insect cells. J Biosci Bioeng 90:508–514

    Article  CAS  PubMed  Google Scholar 

  29. Jaroentomeechai T, Stark JC, Natarajan A, Glasscock CJ, Yates LE, Hsu KJ, Mrksich M, Jewett MC, Delisa MP (2018) Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat Commun 9:1–11

    Google Scholar 

  30. Kightlinger W, Duncker KE, Ramesh A, Thames AH, Natarajan A, Stark JC, Yang A, Lin L, Mrksich M, DeLisa MP et al (2019) A cell-free biosynthesis platform for modular construction of protein glycosylation pathways. Nat Commun 10

    Google Scholar 

  31. Matsuda T, Watanabe S, Kigawa T (2013) Cell-free synthesis system suitable for disulfide-containing proteins. Biochem Biophys Res Commun 431:296–301

    Article  CAS  PubMed  Google Scholar 

  32. Goerke AR, Swartz JR (2009) High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids. Biotechnol Bioeng 102:400–416

    Article  CAS  PubMed  Google Scholar 

  33. Des Soye BJ, Patel JR, Isaacs FJ, Jewett MC (2015) Repurposing the translation apparatus for synthetic biology. Curr Opin Chem Biol 28:83–90

    Article  CAS  Google Scholar 

  34. Yim SS, Johns NI, Park J, Gomes AL, McBee RM, Richardson M, Ronda C, Chen SP, Garenne D, Noireaux V et al (2019) Multiplex transcriptional characterizations across diverse bacterial species using cell-free systems. Mol Syst Biol 15:1–15

    Article  Google Scholar 

  35. Chappell J, Jensen K, Freemont PS (2013) Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology. Nucleic Acids Res 41:3471–3481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Niederholtmeyer H, Sun ZZ, Hori Y, Yeung E, Verpoorte A, Murray RM, Maerkl SJ (2015) Rapid cell-free forward engineering of novel genetic ring oscillators. elife 4:1–18

    Article  Google Scholar 

  37. Karim AS, Jewett MC (2016) A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery. Metab Eng 36:116–126

    Article  CAS  PubMed  Google Scholar 

  38. Dudley QM, Anderson KC, Jewett MC (2016) Cell-free mixing of Escherichia coli crude extracts to prototype and rationally engineer high-titer mevalonate synthesis. ACS Synth Biol 5:1578–1588

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Silverman AD, Karim AS, Jewett MC (2020) Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet 21:151–170

    Article  CAS  PubMed  Google Scholar 

  40. Voloshin AM, Swartz JR (2008) Large-scale batch reactions for cell-free protein synthesis. In: Cell-free protein synthesis

    Google Scholar 

  41. Zawada JF, Yin G, Steiner AR, Yang J, Naresh A, Roy SM, Gold DS, Heinsohn HG, Murray CJ (2011) Microscale to manufacturing scale-up of cell-free cytokine production-a new approach for shortening protein production development timelines. Biotechnol Bioeng 108:1570–1578

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Pardee K, Green AA, Ferrante T, Cameron DE, Daleykeyser A, Yin P, Collins JJ (2014) Paper-based synthetic gene networks. Cell 159:940–954

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Pardee K, Green AA, Takahashi MK, Braff D, Lambert G, Lee JW, Ferrante T, Ma D, Donghia N, Fan M et al (2016) Rapid, low-cost detection of zika virus using programmable biomolecular components. Cell 165:1255–1266

    Article  CAS  PubMed  Google Scholar 

  44. Ma D, Shen L, Wu K, Diehnelt CW, Green AA (2018) Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment. Synth Biol 3:1–11

    Article  CAS  Google Scholar 

  45. Voyvodic PL, Pandi A, Koch M, Conejero I, Valjent E, Courtet P, Renard E, Faulon JL, Bonnet J (2019) Plug-and-play metabolic transducers expand the chemical detection space of cell-free biosensors. Nat Commun 10:1–8

    Article  CAS  Google Scholar 

  46. Gräwe A, Dreyer A, Vornholt T, Barteczko U, Buchholz L, Drews G, Ho UL, Jackowski ME, Kracht M, Lüders J et al (2019) A paper-based, cell-free biosensor system for the detection of heavy metals and date rape drugs. PLoS One 14:1–22

    Article  Google Scholar 

  47. Stark JC, Jaroentomeechai T, Moeller TD, Hershewe JM, Warfel KF, Moricz BS, Martini AM, Dubner RS, Hsu KJ, Stevenson TC et al (2021) On-demand biomanufacturing of protective conjugate vaccines. Sci Adv 7

    Google Scholar 

  48. Amalfitano E, Karlikow M, Norouzi M, Jaenes K, Cicek S, Masum F, Sadat Mousavi P, Guo Y, Tang L, Sydor A et al (2021) A glucose meter interface for point-of-care gene circuit-based diagnostics. Nat Commun 12:1–10

    Article  Google Scholar 

  49. Burgenson D, Gurramkonda C, Pilli M, Ge X, Andar A, Kostov Y, Tolosa L, Rao G (2018) Rapid recombinant protein expression in cell-free extracts from human blood. Sci Rep 8:1–7

    Article  CAS  Google Scholar 

  50. Kung H, Redfield B, Treadwell BV, Eskin B, Spears C, Weissbach H (1977) DNA-directed in vitro synthesis of β-galactosidase. Studies with purified factors. J Biol Chem 252:6889–6894

    Article  CAS  PubMed  Google Scholar 

  51. Ganoza MC, Cunningham C, Green RM (1985) Isolation and point of action of a factor from Escherichia coli required to reconstruct translation. Proc Natl Acad Sci U S A 82:1648–1652

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Pavlov MY, Ehrenberg M (1996) Rate of translation of natural mRNAs in an optimized in vitro system. Arch Biochem Biophys 328:9–16

    Article  CAS  PubMed  Google Scholar 

  53. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19:751–755

    Article  CAS  PubMed  Google Scholar 

  54. Matsuura T, Kazuta Y, Aita T, Adachi J, Yomo T (2009) Quantifying epistatic interactions among the components constituting the protein translation system. Mol Syst Biol 5:1–10

    Article  Google Scholar 

  55. Doerr A, De Reus E, Van Nies P, Van Der Haar M, Wei K, Kattan J, Wahl A, Danelon C (2019) Modelling cell-free RNA and protein synthesis with minimal systems. Phys Biol 16

    Google Scholar 

  56. Lavickova B, Maerkl SJ (2019) A simple, robust, and low-cost method to produce the PURE cell-free system. ACS Synth Biol 8:455–462

    Article  CAS  PubMed  Google Scholar 

  57. Oberholzer T, Nierhaus KH, Luisi PL (1999) Protein expression in liposomes. Biochem Biophys Res Commun 261:238–241

    Article  CAS  PubMed  Google Scholar 

  58. Yu W, Sato K, Wakabayashi M, Nakaishi T, Ko-Mitamura EP, Shima Y, Urabe I, Yomo T (2001) Synthesis of functional protein in liposome. J Biosci Bioeng 92:590–593

    Article  CAS  PubMed  Google Scholar 

  59. Noireaux V, Libchaber A (2004) A vesicle bioreactor as a step toward an artificial cell assembly. Proc Natl Acad Sci 101:17669–17674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Vogele K, Frank T, Gasser L, Goetzfried MA, Hackl MW, Sieber SA, Simmel FC, Pirzer T (2018) Towards synthetic cells using peptide-based reaction compartments. Nat Commun 9:1–7

    Article  CAS  Google Scholar 

  61. Frank T, Vogele K, Dupin A, Simmel FC, Pirzer T (2020) Growth of giant peptide vesicles driven by compartmentalized transcription–translation activity. Chem – A Eur J 26:17356–17360

    Article  CAS  Google Scholar 

  62. Schreiber A, Huber MC, Schiller SM (2019) Prebiotic protocell model based on dynamic protein membranes accommodating anabolic reactions. Langmuir 35:9593–9610

    Article  CAS  PubMed  Google Scholar 

  63. Huang X, Li M, Green DC, Williams DS, Patil AJ, Mann S (2013) Interfacial assembly of protein–polymer nano-conjugates into stimulus-responsive biomimetic protocells. Nat Commun 4:1–9

    Article  Google Scholar 

  64. Sharma B, Ma Y, Hiraki HL, Baker BM, Ferguson AL, Liu AP (2021) Facile formation of giant elastin-like polypeptide vesicles as synthetic cells. Chem Commun 57:13202–13205

    Article  CAS  Google Scholar 

  65. Martino C, Kim SH, Horsfall L, Abbaspourrad A, Rosser SJ, Cooper J, Weitz DA (2012) Protein expression, aggregation, and triggered release from polymersomes as artificial cell-like structures. Angew Chem Int Ed 51:6416–6420

    Article  CAS  Google Scholar 

  66. Li M, Green DC, Anderson JLR, Binks BP, Mann S (2011) In vitro gene expression and enzyme catalysis in bio-inorganic protocells. Chem Sci 2:1739

    Article  CAS  Google Scholar 

  67. Jiao Y, Liu Y, Luo D, Huck WTS, Yang D (2018) Microfluidic-assisted fabrication of clay microgels for cell-free protein synthesis. ACS Appl Mater Interfaces 10:29308–29313

    Article  CAS  PubMed  Google Scholar 

  68. Yang D, Peng S, Hartman MR, Gupton-Campolongo T, Rice EJ, Chang AK, Gu Z, Lu GQ, Luo D (2013) Enhanced transcription and translation in clay hydrogel and implications for early life evolution. Sci Rep 3:1–6

    Article  CAS  Google Scholar 

  69. Liu J, Guo Z, Li Y, Liang J, Xue J, Xu J, Whitelock JM, Xie L, Kong B, Liang K (2021) pH-gated activation of gene transcription and translation in biocatalytic metal–organic framework artificial cells. Adv NanoBiomed Res 1:2000034

    Article  CAS  Google Scholar 

  70. Tang T-YD, van Swaay D, DeMello A, Ross Anderson JL, Mann S (2015) In vitro gene expression within membrane-free coacervate protocells. Chem Commun 51:11429–11432

    Article  Google Scholar 

  71. Deng N, Huck WTS (2017) Microfluidic formation of monodisperse coacervate organelles in liposomes. Angew Chem 129:9868–9872

    Article  Google Scholar 

  72. Whitfield CJ, Banks AM, Dura G, Love J, Fieldsend JE, Goodchild SA, Fulton DA, Howard TP (2020) Cell-free protein synthesis in hydrogel materials. Chem Commun 56:7108–7111

    Article  CAS  Google Scholar 

  73. Thiele J, Ma Y, Foschepoth D, Hansen MMK, Steffen C, Heus HA, Huck WTS (2014) DNA-functionalized hydrogels for confined membrane-free in vitro transcription/translation. Lab Chip 14:2651

    Article  CAS  PubMed  Google Scholar 

  74. Heida T, Köhler T, Kaufmann A, Männel MJ, Thiele J (2020) Cell-free protein synthesis in bifunctional hyaluronan microgels: a strategy for in situ immobilization and purification of his-tagged proteins. ChemSystemsChem 2:1–7

    Google Scholar 

  75. Köhler T, Heida T, Hoefgen S, Weigel N, Valiante V, Thiele J (2020) Cell-free protein synthesis and: in situ immobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion. RSC Adv 10:40588–40596

    Article  PubMed  PubMed Central  Google Scholar 

  76. Kahn JS, Ruiz RCH, Sureka S, Peng S, Derrien TL, An D, Luo D (2016) DNA microgels as a platform for cell-free protein expression and display. Biomacromolecules 17:2019–2026

    Article  CAS  PubMed  Google Scholar 

  77. Zhou X, Wu H, Cui M, Lai SN, Zheng B (2018) Long-lived protein expression in hydrogel particles: towards artificial cells. Chem Sci 9:4275–4279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Lai SN, Zhou X, Ouyang X, Zhou H, Liang Y, Xia J, Zheng B (2020) Artificial cells capable of long-lived protein synthesis by using aptamer grafted polymer hydrogel. ACS Synth Biol 9:76–83

    Article  CAS  PubMed  Google Scholar 

  79. Strahl H, Errington J (2017) Bacterial membranes: structure, domains, and function. Annu Rev Microbiol 71:519–538

    Article  CAS  PubMed  Google Scholar 

  80. De Gier J, Mandersloot JG, Van Deenen LLM (1968) Lipid composition and permeability of liposomes. Biochim Biophys Acta Biomembr 150:666–675

    Article  Google Scholar 

  81. Reeves JP, Dowben RM (1969) Formation and properties of thin-walled phospholipid vesicles. J Cell Physiol 73:49–60

    Article  CAS  PubMed  Google Scholar 

  82. Horger KS, Estes DJ, Capone R, Mayer M (2009) Films of agarose enable rapid formation of giant liposomes in solutions of physiologic ionic strength. J Am Chem Soc 131:1810–1819

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Weinberger A, Tsai FC, Koenderink GH, Schmidt TF, Itri R, Meier W, Schmatko T, Schröder A, Marques C (2013) Gel-assisted formation of giant unilamellar vesicles. Biophys J 105:154–164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Tsumoto K, Matsuo H, Tomita M, Yoshimura T (2009) Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar. Colloids Surf B Biointerfaces 68:98–105

    Article  CAS  PubMed  Google Scholar 

  85. Taylor P, Xu C, Fletcher PDI, Paunov VN (2003) A novel technique for preparation of monodisperse giant liposomes. Chem Commun 3:1732

    Article  Google Scholar 

  86. Pott T, Bouvrais H, Méléard P (2008) Giant unilamellar vesicle formation under physiologically relevant conditions. Chem Phys Lipids 154:115–119

    Article  CAS  PubMed  Google Scholar 

  87. Moga A, Yandrapalli N, Dimova R, Robinson T (2019) Optimization of the inverted emulsion method for high-yield production of biomimetic giant unilamellar vesicles. Chembiochem 20:2674–2682

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Pautot S, Frisken BJ, Weitz DA (2003) Production of unilamellar vesicles using an inverted emulsion. Langmuir 19:2870–2879

    Article  CAS  Google Scholar 

  89. Van de Cauter L, Fanalista F, van Buren L, De Franceschi N, Godino E, Bouw S, Danelon C, Dekker C, Koenderink GH, Ganzinger KA (2021) Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles. ACS Synth Biol 10:1690–1702

    Article  PubMed  PubMed Central  Google Scholar 

  90. Deshpande S, Caspi Y, Meijering AEC, Dekker C (2016) Octanol-assisted liposome assembly on chip. Nat Commun 7:1–9

    Article  CAS  Google Scholar 

  91. Yandrapalli N, Petit J, Bäumchen O, Robinson T (2020) Surfactant-free production of biomimetic artificial cells using PDMS-based microfluidics. bioRxiv. https://doi.org/10.1101/2020.10.23.346932

  92. Kaltenbach M, Devenish SRA, Hollfelder F (2012) A simple method to evaluate the biochemical compatibility of oil/surfactant mixtures for experiments in microdroplets. Lab Chip 12:4185–4192

    Article  CAS  PubMed  Google Scholar 

  93. Griffiths AD, Tawfik DS (1998) Man-made cell-like compartments for molecular evolution. Nat Biotechnol 16:652–656

    Article  PubMed  Google Scholar 

  94. Chowdhury MS, Zheng W, Kumari S, Heyman J, Zhang X, Dey P, Weitz DA, Haag R (2019) Dendronized fluorosurfactant for highly stable water-in-fluorinated oil emulsions with minimal inter-droplet transfer of small molecules. Nat Commun 10

    Google Scholar 

  95. Syeda R, Holden MA, Hwang WL, Bayley H (2008) Screening blockers against a potassium channel with a droplet interface bilayer array. J Am Chem Soc 130:15543–15548

    Article  CAS  PubMed  Google Scholar 

  96. Friddin MS, Morgan H, de Planque MRR (2013) Cell-free protein expression systems in microdroplets: stabilization of interdroplet bilayers. Biomicrofluidics 7

    Google Scholar 

  97. Booth MJ, Schild VR, Graham AD, Olof SN, Bayley H (2016) Light-activated communication in synthetic tissues. Sci Adv 2:1–12

    Article  Google Scholar 

  98. Rideau E, Dimova R, Schwille P, Wurm FR, Landfester K (2018) Liposomes and polymersomes: a comparative review towards cell mimicking. Chem Soc Rev 47:8572–8610

    Article  CAS  PubMed  Google Scholar 

  99. Kamat NP, Katz JS, Hammer DA (2011) Engineering polymersome protocells. J Phys Chem Lett 2:1612–1623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Discher BM, Won Y, Ege DS, Lee JC, Bates FS, Discher DE, Hammer DA (1999) Polymersomes: tough vesicles made from diblock copolymers. Science 284:1143–1146

    Article  CAS  PubMed  Google Scholar 

  101. Battaglia G, Ryan AJ (2005) Bilayers and interdigitation in block copolymer vesicles. J Am Chem Soc 127:8757–8764

    Article  CAS  PubMed  Google Scholar 

  102. So S, Lodge TP (2014) Rate of molecular exchange through the membranes of ionic liquid filled polymersomes dispersed in water. J Phys Chem C 118:21140–21147

    Article  CAS  Google Scholar 

  103. Nallani M, Andreasson-Ochsner M, Tan C-WD, Sinner E-K, Wisantoso Y, Geifman-Shochat S, Hunziker W (2011) Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes. Biointerphases 6:153

    Article  CAS  PubMed  Google Scholar 

  104. May S, Andreasson-Ochsner M, Fu Z, Low YX, Tan D, De Hoog HPM, Ritz S, Nallani M, Sinner EK (2013) In vitro expressed GPCR inserted in polymersome membranes for ligand-binding studies. Angew Chem Int Ed 52:749–753

    Article  CAS  Google Scholar 

  105. Jacobs ML, Boyd MA, Kamat NP (2019) Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression. Proc Natl Acad Sci U S A 116:4031–4036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Blanken D, Foschepoth D, Serrão AC, Danelon C (2020) Genetically controlled membrane synthesis in liposomes. Nat Commun 11:1–13

    Article  Google Scholar 

  107. Scott A, Noga MJ, De Graaf P, Westerlaken I, Yildirim E, Danelon C (2016) Cell-free phospholipid biosynthesis by gene-encoded enzymes reconstituted in liposomes. PLoS One 11:1–23

    Article  Google Scholar 

  108. Eto S, Matsumura R, Shimane Y, Fujimi M, Berhanu S, Kasama T, Kuruma Y (2022) Phospholipid synthesis inside phospholipid membrane vesicles. Commun Biol 5:1–11

    Article  Google Scholar 

  109. Bhattacharya A, Cho CJ, Brea RJ, Devaraj NK (2021) Expression of fatty acyl-CoA ligase drives one-pot de novo synthesis of membrane-bound vesicles in a cell-free transcription-translation system. J Am Chem Soc 143:11235–11242

    Article  CAS  PubMed  Google Scholar 

  110. Robinson T, Kuhn P, Eyer K, Dittrich PS (2013) Microfluidic trapping of giant unilamellar vesicles to study transport through a membrane pore. Biomicrofluidics 7:044105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Peruzzi JA, Jacobs ML, Vu TQ, Wang KS, Kamat NP (2019) Barcoding biological reactions with DNA-functionalized vesicles. Angew Chem 131:18856–18863

    Article  Google Scholar 

  112. Adamala KP, Martin-Alarcon DA, Guthrie-Honea KR, Boyden ES (2017) Engineering genetic circuit interactions within and between synthetic minimal cells. Nat Chem 9:431–439

    Article  CAS  PubMed  Google Scholar 

  113. De Boer PAJ, Crossley RE, Rothfield LI (1990) Central role for the Escherichia coli minC gene product in two different cell division-inhibition systems. Proc Natl Acad Sci U S A 87:1129–1133

    Article  PubMed  PubMed Central  Google Scholar 

  114. Hu Z, Mukherjee A, Pichoff S, Lutkenhaus J (1999) The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization. Proc Natl Acad Sci U S A 96:14819–14824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. De Boer PAJ, Crossley RE, Rothfield LI (1992) Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli. J Bacteriol 174:63–70

    Article  PubMed  PubMed Central  Google Scholar 

  116. Loose M, Fischer-Friedrich E, Ries J, Kruse K, Schwille P (2008) Spatial regulators for bacterial cell division self-organize into surface waves in vitro. Science 320:789–792

    Article  CAS  PubMed  Google Scholar 

  117. Osawa M, Anderson DE, Erickson HP (2008) Reconstitution of contractile FtsZ rings in liposomes. Science 320:792–794

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Godino E, López JN, Foschepoth D, Cleij C, Doerr A, Castellà CF, Danelon C (2019) De novo synthesized min proteins drive oscillatory liposome deformation and regulate FtsA-FtsZ cytoskeletal patterns. Nat Commun 10:4969

    Article  PubMed  PubMed Central  Google Scholar 

  119. Godino E, Doerr A, Danelon C (2022) Min waves without MinC can pattern FtsA-anchored FtsZ filaments on model membranes. Commun Biol 5:1–13

    Article  Google Scholar 

  120. Godino E, López JN, Zarguit I, Doerr A, Jimenez M, Rivas G, Danelon C (2020) Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes. Commun Biol 3:1–11

    Article  Google Scholar 

  121. Kattan J, Doerr A, Dogterom M, Danelon C (2021) Shaping liposomes by cell-free expressed bacterial microtubules. ACS Synth Biol 10:2447–2455

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Dreher Y, Jahnke K, Bobkova E, Spatz JP, Göpfrich K (2021) Division and regrowth of phase-separated giant unilamellar vesicles**. Angew Chem 133:10756–10764

    Article  Google Scholar 

  123. Steinkühler J, Knorr RL, Zhao Z, Bhatia T, Bartelt SM, Wegner S, Dimova R, Lipowsky R (2020) Controlled division of cell-sized vesicles by low densities of membrane-bound proteins. Nat Commun 11:905

    Article  PubMed  PubMed Central  Google Scholar 

  124. Kurokawa C, Fujiwara K, Morita M, Kawamata I, Kawagishi Y, Sakai A, Murayama Y, Nomura SM, Murata S, Takinoue M et al (2017) DNA cytoskeleton for stabilizing artificial cells. Proc Natl Acad Sci 114:7228–7233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Jahnke K, Huth V, Mersdorf U, Liu N, Göpfrich K (2021) Bottom-up assembly of synthetic cells with a DNA cytoskeleton. ACS Nano. https://doi.org/10.1021/acsnano.1c10703

  126. Litschel T, Kelley CF, Holz D, Adeli Koudehi M, Vogel SK, Burbaum L, Mizuno N, Vavylonis D, Schwille P (2021) Reconstitution of contractile actomyosin rings in vesicles. Nat Commun 12:2254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Elowitz MB, Levine AJ, Siggia ED, Swain PS (2002) Stochastic gene expression in a single cell. Science 297:1183–1186

    Article  CAS  PubMed  Google Scholar 

  128. Bar-Even A, Paulsson J, Maheshri N, Carmi M, O’Shea E, Pilpel Y, Barkai N (2006) Noise in protein expression scales with natural protein abundance. Nat Genet 38:636–643

    Article  CAS  PubMed  Google Scholar 

  129. Hansen MMK, Meijer LHH, Spruijt E, Maas RJM, Rosquelles MV, Groen J, Heus HA, Huck WTS (2015) Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets. Nat Nanotechnol 11:191–197

    Article  PubMed  PubMed Central  Google Scholar 

  130. Nishimura K, Tsuru S, Suzuki H, Yomo T (2015) Stochasticity in gene expression in a cell-sized compartment. ACS Synth Biol 4:566–576

    Article  CAS  PubMed  Google Scholar 

  131. Wang S, Majumder S, Emery NJ, Liu AP (2018) Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets. Synth Biol 3:1–9

    Article  Google Scholar 

  132. Sakamoto R, Noireaux V, Maeda YT (2018) Anomalous scaling of gene expression in confined cell-free reactions. Sci Rep 8:7364

    Article  PubMed  PubMed Central  Google Scholar 

  133. Gonzales DT, Yandrapalli N, Robinson T, Zechner C, T-YD T (2022) Cell-free gene expression dynamics in synthetic cell populations. ACS Synth Biol 11:205–215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Vibhute MA, Schaap MH, Maas RJM, Nelissen FHT, Spruijt E, Heus HA, Hansen MMK, Huck WTS (2020) Transcription and translation in cytomimetic protocells perform most efficiently at distinct macromolecular crowding conditions. ACS Synth Biol 9:2797–2807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Deng NN, Vibhute MA, Zheng L, Zhao H, Yelleswarapu M, Huck WTS (2018) Macromolecularly crowded protocells from reversibly shrinking monodisperse liposomes. J Am Chem Soc 140:7399–7402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Matthies D, Haberstock S, Joos F, Dötsch V, Vonck J, Bernhard F, Meier T (2011) Cell-free expression and assembly of ATP synthase. J Mol Biol 413:593–603

    Article  CAS  PubMed  Google Scholar 

  137. Berhanu S, Ueda T, Kuruma Y (2019) Artificial photosynthetic cell producing energy for protein synthesis. Nat Commun 10

    Google Scholar 

  138. Sakatani Y, Yomo T, Ichihashi N (2018) Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination. Sci Rep 8:1–5

    Article  CAS  Google Scholar 

  139. Okauchi H, Ichihashi N (2021) Continuous cell-free replication and evolution of artificial genomic DNA in a compartmentalized gene expression system. ACS Synth Biol 10:3507–3517

    Article  CAS  PubMed  Google Scholar 

  140. Van Nies P, Westerlaken I, Blanken D, Salas M, Mencía M, Danelon C (2018) Self-replication of DNA by its encoded proteins in liposome-based synthetic cells. Nat Commun 9:1–12

    Google Scholar 

  141. Libicher K, Hornberger R, Heymann M, Mutschler H (2020) In vitro self-replication and multicistronic expression of large synthetic genomes. Nat Commun 11:1–8

    Article  Google Scholar 

  142. Fujii R, Kitaoka M, Hayashi K (2006) Error-prone rolling circle amplification: the simplest random mutagenesis protocol. Nat Protoc 1:2493–2497

    Article  CAS  PubMed  Google Scholar 

  143. Kita H, Matsuura T, Sunami T, Hosoda K, Ichihashi N, Tsukada K, Urabe I, Yomo T (2008) Replication of genetic information with self-encoded replicase in liposomes. Chembiochem 9:2403–2410

    Article  CAS  PubMed  Google Scholar 

  144. Mizuuchi R, Ichihashi N (2020) Translation-coupled RNA replication and parasitic replicators in membrane-free compartments. Chem Commun 56:13453–13456

    Article  CAS  Google Scholar 

  145. Caschera F, Karim AS, Gazzola G, D’Aquino AE, Packard NH, Jewett MC (2018) High-throughput optimization cycle of a cell-free ribosome assembly and protein synthesis system. ACS Synth Biol 7:2841–2853

    Article  CAS  PubMed  Google Scholar 

  146. Caschera F, Lee JW, Ho KKY, Liu AP, Jewett MC (2016) Cell-free compartmentalized protein synthesis inside double emulsion templated liposomes with in vitro synthesized and assembled ribosomes. Chem Commun 52:5467–5469

    Article  CAS  Google Scholar 

  147. Niederholtmeyer H, Chaggan C, Devaraj NK (2018) Communication and quorum sensing in non-living mimics of eukaryotic cells. Nat Commun 9:5027

    Article  PubMed  PubMed Central  Google Scholar 

  148. Dupin A, Simmel FC (2019) Signalling and differentiation in emulsion-based multi-compartmentalized in vitro gene circuits. Nat Chem 11:32–39

    Article  CAS  PubMed  Google Scholar 

  149. Dupin A, Aufinger L, Styazhkin I, Rothfischer F, Kaufmann BK, Schwarz S, Galensowske N, Clausen-Schaumann H, Simmel FC (2022) Synthetic cell–based materials extract positional information from morphogen gradients. Sci Adv 8:1–19

    Article  Google Scholar 

  150. Jung K, Fabiani F, Hoyer E, Lassak J (2018) Bacterial transmembrane signalling systems and their engineering for biosensing. Open Biol 8

    Google Scholar 

  151. Peruzzi JA, Steinkühler J, Vu TQ, Gunnels TF, Lu P, Baker D, Kamat NP (2022) Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes. bioRxiv. https://doi.org/10.1101/2022.06.01.494374

  152. Steinküher J, Peruzzi JA, Krüger A, Jacobs ML, Michael C (2023) Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation. bioRxiv. https://doi.org/10.1101/2023.02.10.527944

  153. Peruzzi JA, Galvez NR, Kamat NP (2022) Engineering transmembrane signal transduction in synthetic membranes using two-component systems. bioRxiv

    Google Scholar 

  154. Jayaraman P, Yeoh JW, Jayaraman S, Teh AY, Zhang J, Poh CL (2018) Cell-free optogenetic gene expression system. ACS Synth Biol 7:986–994

    Article  CAS  PubMed  Google Scholar 

  155. Adir O, Albalak MR, Abel R, Weiss LE, Chen G, Gruber A, Staufer O, Kurman Y, Kaminer I, Shklover J et al (2022) Synthetic cells with self-activating optogenetic proteins communicate with natural cells. Nat Commun 13:2328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. Krinsky N, Kaduri M, Zinger A, Shainsky-Roitman J, Goldfeder M, Benhar I, Hershkovitz D, Schroeder A (2018) Synthetic cells synthesize therapeutic proteins inside tumors. Adv Healthc Mater 7:1701163

    Article  Google Scholar 

  157. Lussier F, Staufer O, Platzman I, Spatz JP (2021) Can bottom-up synthetic biology generate advanced drug-delivery systems? Trends Biotechnol 39:445–459

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    David T. Gonzales, Surased Suraritdechachai & T. -Y. Dora Tang

  2. Center for Systems Biology Dresden, Dresden, Germany

    David T. Gonzales & T. -Y. Dora Tang

  3. Physics of Life, Cluster of Excellence, TU Dresden, Dresden, Germany

    T. -Y. Dora Tang

Authors
  1. David T. Gonzales
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Surased Suraritdechachai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. -Y. Dora Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T. -Y. Dora Tang .

Editor information

Editors and Affiliations

  1. Department of Chemical Engineering, Tsinghua University, Beijing, China

    Yuan Lu

  2. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Michael C. Jewett

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Nature Switzerland AG

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Gonzales, D.T., Suraritdechachai, S., Tang, T.Y.D. (2023). Compartmentalized Cell-Free Expression Systems for Building Synthetic Cells. In: Lu, Y., Jewett, M.C. (eds) Cell-free Production. Advances in Biochemical Engineering/Biotechnology, vol 186. Springer, Cham. https://doi.org/10.1007/10_2023_221

Download citation

Publish with us

Policies and ethics

Search

Navigation