{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T00:13:00Z","timestamp":1768522380373,"version":"3.49.0"},"reference-count":35,"publisher":"Springer Science and Business Media LLC","issue":"S7","license":[{"start":{"date-parts":[[2013,4,1]],"date-time":"2013-04-01T00:00:00Z","timestamp":1364774400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/2.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Bioinformatics"],"published-print":{"date-parts":[[2013,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>The wet-lab synthesis of the simplest forms of life (minimal cells) is a challenging aspect in modern synthetic biology. Quasi-cellular systems able to produce proteins directly from DNA can be obtained by encapsulating the cell-free transcription\/translation system PURESYSTEM\u2122(PS) in liposomes. It is possible to detect the intra-vesicle protein production using DNA encoding for GFP and monitoring the fluorescence emission over time. The entrapment of solutes in small-volume liposomes is a fundamental open problem. Stochastic simulation is a valuable tool in the study of biochemical reaction at nanoscale range. QDC (Quick Direct-Method Controlled), a stochastic simulation software based on the well-known Gillespie's SSA algorithm, was used. A suitable model formally describing the PS reactions network was developed, to predict, from inner species concentrations (very difficult to measure in small-volumes), the resulting fluorescence signal (experimentally observable).<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>Thanks to suitable features specific of QDC, we successfully formalized the dynamical coupling between the transcription and translation processes that occurs in the real PS, thus bypassing the concurrent-only environment of Gillespie's algorithm. Simulations were firstly performed for large liposomes (2.67\u00b5m of diameter) entrapping the PS to synthetize GFP. By varying the initial concentrations of the three main classes of molecules involved in the PS (DNA, enzymes, consumables), we were able to stochastically simulate the time-course of GFP-production. The sigmoid fit of the GFP-production curves allowed us to extract three quantitative parameters which are significantly dependent on the various initial states. Then we extended this study for small-volume liposomes (575 nm of diameter), where it is more complex to infer the intra-vesicle composition, due to the expected anomalous entrapment phenomena. We identified almost two extreme states that are forecasted to give rise to significantly different experimental observables.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>The present work is the first one describing in the detail the stochastic behavior of the PS. Thanks to our results, an experimental approach is now possible, aimed at recording the GFP production kinetics in very small micro-emulsion droplets or liposomes, and inferring, by using the simulation as a reverse-engineering procedure, the internal solutes distribution, and shed light on the still unknown forces driving the entrapment phenomenon.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1471-2105-14-s7-s7","type":"journal-article","created":{"date-parts":[[2019,12,11]],"date-time":"2019-12-11T02:00:20Z","timestamp":1576029620000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["Quasi-cellular systems: stochastic simulation analysis at nanoscale range"],"prefix":"10.1186","volume":"14","author":[{"given":"Lorenzo","family":"Calviello","sequence":"first","affiliation":[]},{"given":"Pasquale","family":"Stano","sequence":"additional","affiliation":[]},{"given":"Fabio","family":"Mavelli","sequence":"additional","affiliation":[]},{"given":"Pier Luigi","family":"Luisi","sequence":"additional","affiliation":[]},{"given":"Roberto","family":"Marangoni","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2013,4,22]]},"reference":[{"issue":"6818","key":"5817_CR1","doi-asserted-by":"publisher","first-page":"387","DOI":"10.1038\/35053176","volume":"409","author":"JW Szostak","year":"2001","unstructured":"Szostak JW, Bartel DP, Luisi PL: Synthesizing life. Nature. 2001, 409 (6818): 387-390. 10.1038\/35053176.","journal-title":"Nature"},{"issue":"(9140d519-fc79-","key":"5817_CR2","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/msb4100090","volume":"2","author":"A Forster","year":"2006","unstructured":"Forster A, Church G: Towards synthesis of a minimal cell. Molecular systems biology. 2006, 2 ((9140d519-fc79-e312-0a8a-bfb7cec33a4e): 45.","journal-title":"Molecular systems biology"},{"issue":"47","key":"5817_CR3","doi-asserted-by":"publisher","first-page":"18887","DOI":"10.1039\/c1jm12298c","volume":"21","author":"P Stano","year":"2011","unstructured":"Stano P, Carrara P, Kuruma Y, Pereira de Souza T, Luisi PL: Compartmentalized reactions as a case of soft-matter biotechnology: synthesis of proteins and nucleic acids inside lipid vesicles. Journal of Materials Chemistry. 2011, 21 (47): 18887-18902. 10.1039\/c1jm12298c.","journal-title":"Journal of Materials Chemistry"},{"issue":"e975186c-6d35-e","key":"5817_CR4","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/s00114-005-0056-z","volume":"93","author":"P Luisi","year":"2006","unstructured":"Luisi P, Ferri F, Stano P: Approaches to semi-synthetic minimal cells: a review. Naturwissenschaften. 2006, 93 (e975186c-6d35-e5a8-7236-239c98c5ea6f): 1-14.","journal-title":"Naturwissenschaften"},{"issue":"17","key":"5817_CR5","doi-asserted-by":"publisher","first-page":"7541","DOI":"10.1021\/ja00096a010","volume":"116","author":"P Walde","year":"1994","unstructured":"Walde P, Goto A, Monnard P-A, Wessicken M, Luisi PL: Oparin's Reactions Revisited: Enzymic Synthesis of Poly(adenylic acid) in Micelles and Self-Reproducing Vesicles. Journal of the American Chemical Society. 1994, 116 (17): 7541-7547. 10.1021\/ja00096a010.","journal-title":"Journal of the American Chemical Society"},{"issue":"10","key":"5817_CR6","doi-asserted-by":"publisher","first-page":"677","DOI":"10.1016\/1074-5521(95)90031-4","volume":"2","author":"T Oberholzer","year":"1995","unstructured":"Oberholzer T, Albrizio M, Luisi PL: Polymerase chain reaction in liposomes. Chemistry & biology. 1995, 2 (10): 677-682. 10.1016\/1074-5521(95)90031-4.","journal-title":"Chemistry & biology"},{"issue":"2","key":"5817_CR7","doi-asserted-by":"publisher","first-page":"238","DOI":"10.1006\/bbrc.1999.0404","volume":"261","author":"T Oberholzer","year":"1999","unstructured":"Oberholzer T, Nierhaus KH, Luisi PL: Protein expression in liposomes. Biochem Biophys Res Commun. 1999, 261 (2): 238-241. 10.1006\/bbrc.1999.0404.","journal-title":"Biochem Biophys Res Commun"},{"issue":"6","key":"5817_CR8","doi-asserted-by":"publisher","first-page":"590","DOI":"10.1016\/S1389-1723(01)80322-4","volume":"92","author":"WEI Yu","year":"2001","unstructured":"Yu WEI, Sato K, Wakabayashi M, Nakaishi T, Ko-Mitamura EP, Shima Y, Urabe I, Yomo T: Synthesis of Functional Protein in Liposome. Journal of Bioscience and Bioengineering. 2001, 92 (6): 590-593.","journal-title":"Journal of Bioscience and Bioengineering"},{"issue":"2","key":"5817_CR9","doi-asserted-by":"publisher","first-page":"567","DOI":"10.1016\/j.bbamem.2008.10.017","volume":"1788","author":"Y Kuruma","year":"2009","unstructured":"Kuruma Y, Stano P, Ueda T, Luisi PL: A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells. Biochimica et biophysica acta. 2009, 1788 (2): 567-574. 10.1016\/j.bbamem.2008.10.017.","journal-title":"Biochimica et biophysica acta"},{"issue":"11","key":"5817_CR10","doi-asserted-by":"publisher","first-page":"8544","DOI":"10.1021\/la904569m","volume":"26","author":"T Sunami","year":"2010","unstructured":"Sunami T, Hosoda K, Suzuki H, Matsuura T, Yomo T: Cellular compartment model for exploring the effect of the lipidic membrane on the kinetics of encapsulated biochemical reactions. Langmuir : the ACS journal of surfaces and colloids. 2010, 26 (11): 8544-8595. 10.1021\/la904569m.","journal-title":"Langmuir : the ACS journal of surfaces and colloids"},{"key":"5817_CR11","doi-asserted-by":"crossref","unstructured":"Hosoda K, Sunami T, Kazuta Y, Matsuura T: Quantitative study of the structure of multilamellar giant liposomes as a container of protein synthesis reaction. Langmuir. 2008, 24 (23):","DOI":"10.1021\/la802432f"},{"issue":"10","key":"5817_CR12","doi-asserted-by":"publisher","first-page":"1640","DOI":"10.1002\/cbic.200900205","volume":"10","author":"H Saito","year":"2009","unstructured":"Saito H, Kato Y, Le Berre M, Yamada A, Inoue T, Yosikawa K, Baigl D: Time-resolved tracking of a minimum gene expression system reconstituted in giant liposomes. Chembiochem. 2009, 10 (10): 1640-1643. 10.1002\/cbic.200900205.","journal-title":"Chembiochem"},{"issue":"14","key":"5817_CR13","doi-asserted-by":"publisher","first-page":"1989","DOI":"10.1002\/cbic.201000381","volume":"11","author":"P Luisi","year":"2010","unstructured":"Luisi P, Allegretti M, Pereira de Souza T, Steiniger F, Fahr A, Stano P: Spontaneous protein crowding in liposomes: a new vista for the origin of cellular metabolism. Chembiochem : a European journal of chemical biology. 2010, 11 (14): 1989-2081. 10.1002\/cbic.201000381.","journal-title":"Chembiochem : a European journal of chemical biology"},{"key":"5817_CR14","volume-title":"Chembiochem : a European journal of chemical biology","author":"T Pereira de Souza","year":"2011","unstructured":"Pereira de Souza T, Steiniger F, Stano P, Fahr A, Luisi P: Spontaneous Crowding of Ribosomes and Proteins inside Vesicles: A Possible Mechanism for the Origin of Cell Metabolism. Chembiochem : a European journal of chemical biology. 2011, (2c6f5d5a-f6b1-7d40-4922-239c9858e542)"},{"key":"5817_CR15","volume-title":"Nature","author":"Y Shimizu","year":"2001","unstructured":"Shimizu Y, Inoue A, Tomari Y, Suzuki T: Cell-free translation reconstituted with purified components. Nature. 2001, (28f9cb18-d3a3-328d-ae54-239c98cdc21d)"},{"issue":"4","key":"5817_CR16","doi-asserted-by":"publisher","first-page":"439","DOI":"10.1007\/s11084-007-9086-1","volume":"37","author":"P Stano","year":"2007","unstructured":"Stano P: Question 7: New Aspects of Interactions Among Vesicles. Origins of Life and Evolution of Biospheres. 2007, 37 (4): 439-444. 10.1007\/s11084-007-9086-1.","journal-title":"Origins of Life and Evolution of Biospheres"},{"issue":"1","key":"5817_CR17","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1016\/j.biosystems.2012.01.002","volume":"109","author":"P Stano","year":"2012","unstructured":"Stano P, Rampioni G, Carrara P, Damiano L, Leoni L, Luisi PL: Semi-synthetic minimal cells as a tool for biochemical ICT. Bio Systems. 2012, 109 (1): 24-34. 10.1016\/j.biosystems.2012.01.002.","journal-title":"Bio Systems"},{"key":"5817_CR18","unstructured":"Pereira de Souza T, Stano P, Luisi PL: The Minimal Size of Liposome-Based Model Cells Brings about a Remarkably Enhanced Entrapment and Protein Synthesis. ChemBioChem. 2009, 10 (aaece635-437c-a62d-75d9-239c9855d981):"},{"key":"5817_CR19","unstructured":"Pereira de Souza P, Steiniger F, Stano P, Fahr A, Luisi PL: Spontaneous Crowding of Ribosomes and Proteins inside Vesicles: A Possible Mechanism for the Origin of Cell Metabolism. ChemBioChem. 2011, 12 (2c6f5d5a-f6b1-7d40-4922-239c9858e542):"},{"issue":"Suppl 4","key":"5817_CR20","doi-asserted-by":"publisher","first-page":"S10","DOI":"10.1186\/1471-2105-13-S4-S10","volume":"13","author":"F Mavelli","year":"2012","unstructured":"Mavelli F: Stochastic simulations of minimal cells: the Ribocell model. BMC bioinformatics. 2012, 13 (Suppl 4): S10-10.1186\/1471-2105-13-S4-S10.","journal-title":"BMC bioinformatics"},{"key":"5817_CR21","doi-asserted-by":"publisher","first-page":"S9","DOI":"10.1186\/1471-2105-13-S4-S9","volume":"13","author":"L Lazzerini-Ospri","year":"2012","unstructured":"Lazzerini-Ospri L, Stano P, Luisi P, Marangoni R: Characterization of the emergent properties of a synthetic quasi-cellular system. BMC bioinformatics. 2012, 13: S9.","journal-title":"BMC bioinformatics"},{"key":"5817_CR22","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1146\/annurev.physchem.58.032806.104637","volume":"58","author":"DT Gillespie","year":"2007","unstructured":"Gillespie DT: Stochastic simulation of chemical kinetics. Annual review of physical chemistry. 2007, 58: 35-55. 10.1146\/annurev.physchem.58.032806.104637.","journal-title":"Annual review of physical chemistry"},{"issue":"1-3","key":"5817_CR23","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1016\/j.theochem.2006.03.016","volume":"771","author":"F Mavelli","year":"2006","unstructured":"Mavelli F, Piotto S: Stochastic simulations of homogeneous chemically reacting systems. Journal of Molecular Structure: THEOCHEM. 2006, 771 (1-3): 55-64. 10.1016\/j.theochem.2006.03.016.","journal-title":"Journal of Molecular Structure: THEOCHEM"},{"issue":"4","key":"5817_CR24","doi-asserted-by":"publisher","first-page":"403","DOI":"10.1016\/0021-9991(76)90041-3","volume":"22","author":"DT Gillespie","year":"1976","unstructured":"Gillespie DT: A general method for numerically simulating the stochastic time evolution of coupled chemical reactions. Journal of Computational Physics. 1976, 22 (4): 403-434. 10.1016\/0021-9991(76)90041-3.","journal-title":"Journal of Computational Physics"},{"issue":"79563a14-1272-f","key":"5817_CR25","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1186\/1471-2105-12-121","volume":"12","author":"J M\u00e4kel\u00e4","year":"2011","unstructured":"M\u00e4kel\u00e4 J, Lloyd-Price J, Yli-Harja O, Ribeiro A: Stochastic sequence-level model of coupled transcription and translation in prokaryotes. BMC bioinformatics. 2011, 12 (79563a14-1272-feb7-481d-85b1f894f09e): 121.","journal-title":"BMC bioinformatics"},{"key":"5817_CR26","unstructured":"Stogbauer T, Windhanger L, Zimmer R, Radler JO: Experiment and mathematical modeling of gene expression dynamics in a cell-free system. Integrative Biology. 2012, 4 (b372eb93-1286-890c-a0d2-94e985aa0a47):"},{"issue":"13","key":"5817_CR27","doi-asserted-by":"publisher","first-page":"8644","DOI":"10.1074\/jbc.272.13.8644","volume":"272","author":"SS Sastry","year":"1997","unstructured":"Sastry SS, Ross BM: Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes. The Journal of biological chemistry. 1997, 272 (13): 8644-8652. 10.1074\/jbc.272.13.8644.","journal-title":"The Journal of biological chemistry"},{"issue":"2","key":"5817_CR28","doi-asserted-by":"publisher","first-page":"365","DOI":"10.1016\/0022-2836(89)90260-X","volume":"207","author":"MA Sorensen","year":"1989","unstructured":"Sorensen MA, Kurland CG, Pedersen S: Codon usage determines translation rate in Escherichia coli. Journal of molecular biology. 1989, 207 (2): 365-377. 10.1016\/0022-2836(89)90260-X.","journal-title":"Journal of molecular biology"},{"issue":"2","key":"5817_CR29","doi-asserted-by":"publisher","first-page":"425","DOI":"10.1073\/pnas.0510013103","volume":"103","author":"JI Glass","year":"2006","unstructured":"Glass JI, Assad-Garcia N, Alperovich N, Yooseph S, Lewis MR, Maruf M, Hutchison CA, Smith HO, Venter JC: Essential genes of a minimal bacterium. Proceedings of the National Academy of Sciences of the United States of America. 2006, 103 (2): 425-430. 10.1073\/pnas.0510013103.","journal-title":"Proceedings of the National Academy of Sciences of the United States of America"},{"issue":"Database","key":"5817_CR30","doi-asserted-by":"publisher","first-page":"D750","DOI":"10.1093\/nar\/gkp889","volume":"38","author":"R Milo","year":"2010","unstructured":"Milo R, Jorgensen P, Moran U, Weber G, Springer M: BioNumbers--the database of key numbers in molecular and cell biology. Nucleic acids research. 2010, 38 (Database): D750-753. 10.1093\/nar\/gkp889.","journal-title":"Nucleic acids research"},{"issue":"5","key":"5817_CR31","doi-asserted-by":"publisher","first-page":"3239","DOI":"10.1074\/jbc.M310471200","volume":"279","author":"GM Skinner","year":"2004","unstructured":"Skinner GM, Baumann CG, Quinn DM, Molloy JE, Hoggett JG: Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle. The Journal of biological chemistry. 2004, 279 (5): 3239-3244.","journal-title":"The Journal of biological chemistry"},{"issue":"3","key":"5817_CR32","doi-asserted-by":"publisher","first-page":"717","DOI":"10.1529\/biophysj.106.087825","volume":"92","author":"H Zouridis","year":"2007","unstructured":"Zouridis H, Hatzimanikatis V: A model for protein translation: polysome self-organization leads to maximum protein synthesis rates. Biophys J. 2007, 92 (3): 717-730. 10.1529\/biophysj.106.087825.","journal-title":"Biophys J"},{"issue":"13","key":"5817_CR33","doi-asserted-by":"publisher","first-page":"2640","DOI":"10.1093\/nar\/25.13.2640","volume":"25","author":"T Kubori","year":"1997","unstructured":"Kubori T, Shimamoto N: Physical interference between Escherichia coli RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation. Nucleic acids research. 1997, 25 (13): 2640-2647. 10.1093\/nar\/25.13.2640.","journal-title":"Nucleic acids research"},{"issue":"2","key":"5817_CR34","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1016\/j.cell.2008.11.016","volume":"136","author":"F Brandt","year":"2009","unstructured":"Brandt F, Etchells SA, Ortiz JO, Elcock AH, Hartl FU, Baumeister W: The native 3D organization of bacterial polysomes. Cell. 2009, 136 (2): 261-271. 10.1016\/j.cell.2008.11.016.","journal-title":"Cell"},{"issue":"4","key":"5817_CR35","doi-asserted-by":"publisher","first-page":"492","DOI":"10.1016\/j.copbio.2009.08.004","volume":"20","author":"C Chiarabelli","year":"2009","unstructured":"Chiarabelli C, Stano P, Luisi PL: Chemical approaches to synthetic biology. Current opinion in biotechnology. 2009, 20 (4): 492-497. 10.1016\/j.copbio.2009.08.004.","journal-title":"Current opinion in biotechnology"}],"container-title":["BMC Bioinformatics"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/link.springer.com\/content\/pdf\/10.1186\/1471-2105-14-S7-S7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"http:\/\/link.springer.com\/article\/10.1186\/1471-2105-14-S7-S7\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/1471-2105-14-S7-S7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,9,1]],"date-time":"2021-09-01T23:17:36Z","timestamp":1630538256000},"score":1,"resource":{"primary":{"URL":"https:\/\/bmcbioinformatics.biomedcentral.com\/articles\/10.1186\/1471-2105-14-S7-S7"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2013,4]]},"references-count":35,"journal-issue":{"issue":"S7","published-print":{"date-parts":[[2013,4]]}},"alternative-id":["5817"],"URL":"https:\/\/doi.org\/10.1186\/1471-2105-14-s7-s7","relation":{},"ISSN":["1471-2105"],"issn-type":[{"value":"1471-2105","type":"electronic"}],"subject":[],"published":{"date-parts":[[2013,4]]},"assertion":[{"value":"22 April 2013","order":1,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}}],"article-number":"S7"}}