Co-generating synthetic parts toward a self-replicating system

Li, Jun; Haas, Wilhelm; Jackson, Kirsten; Kuru, Erkin; Jewett, Michael C.; Fan, Z. Hugh; Gygi, Steven P.; Church, George M.

doi:10.1021/acssynbio.6b00342

Co-generating synthetic parts toward a self-replicating system

Journal Article · Thu Mar 23 00:00:00 EDT 2017 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.6b00342· OSTI ID:1348832

Li, Jun ^[1]; Haas, Wilhelm ^[2]; Jackson, Kirsten ^[3]; Kuru, Erkin ^[4]; Jewett, Michael C. ^[5]; Fan, Z. Hugh ^[3]; Gygi, Steven P. ^[2]; Church, George M. ^[4]

Harvard Medical School, Boston, MA (United States); Wyss Harvard Institute of Biologically Inspired Engineering, Boston, MA (United States); Harvard Medical School, Genetics Department
Harvard Medical School, Boston, MA (United States)
Univ. of Florida, Gainesville, FL (United States)
Harvard Medical School, Boston, MA (United States); Wyss Harvard Institute of Biologically Inspired Engineering, Boston, MA (United States)
Northwestern Univ., Evanston, IL (United States)

To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE, might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when co-expressed in batch format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in co-expression pool varied from ~1.5 to 5-fold compared to the batch mode, with up to ~ 2.4 µM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ~13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. As a result, this work also points to what is still needed to obtain self-replicating synthetic ribosomes in-situ in the PURE system.

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Li, Jun, et al. "Co-generating synthetic parts toward a self-replicating system." ACS Synthetic Biology, vol. 6, no. 7, Mar. 2017. https://doi.org/10.1021/acssynbio.6b00342

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Li, Jun, Haas, Wilhelm, Jackson, Kirsten, Kuru, Erkin, Jewett, Michael C., Fan, Z. Hugh, Gygi, Steven P., & Church, George M. (2017). Co-generating synthetic parts toward a self-replicating system. ACS Synthetic Biology, 6(7). https://doi.org/10.1021/acssynbio.6b00342

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Li, Jun, Haas, Wilhelm, Jackson, Kirsten, et al., "Co-generating synthetic parts toward a self-replicating system," ACS Synthetic Biology 6, no. 7 (2017), https://doi.org/10.1021/acssynbio.6b00342

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@article{osti_1348832,
  author       = {Li, Jun and Haas, Wilhelm and Jackson, Kirsten and Kuru, Erkin and Jewett, Michael C. and Fan, Z. Hugh and Gygi, Steven P. and Church, George M.},
  title        = {Co-generating synthetic parts toward a self-replicating system},
  annote       = {To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE, might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system.  We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when co-expressed in batch format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in co-expression pool varied from ~1.5 to 5-fold compared to the batch mode, with up to ~ 2.4 µM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ~13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. As a result, this work also points to what is still needed to obtain self-replicating synthetic ribosomes in-situ in the PURE system.},
  doi          = {10.1021/acssynbio.6b00342},
  url          = {https://www.osti.gov/biblio/1348832},
  journal      = {ACS Synthetic Biology},
  issn         = {ISSN 2161-5063},
  number       = {7},
  volume       = {6},
  place        = {United States},
  publisher    = {American Chemical Society (ACS)},
  year         = {2017},
  month        = {03}}

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Research Organization:: Harvard Medical School, Boston, MA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: FG02-02ER63445

OSTI ID:: 1348832

Journal Information:: ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Journal Issue: 7 Vol. 6; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (33)

Optimization of a miniaturized fluid array device for cell‐free protein synthesis Jackson, Kirsten; Jin, Shouguang; Fan, Z. Hugh Biotechnology and Bioengineering, Vol. 112, Issue 12 https://doi.org/10.1002/bit.25668	journal	July 2015
PURE Technology Shimizu, Yoshihiro; Ueda, Takuya Methods in Molecular Biology https://doi.org/10.1007/978-1-60327-331-2_2	book	November 2009
A Tissue-Specific Atlas of Mouse Protein Phosphorylation and Expression Huttlin, Edward L.; Jedrychowski, Mark P.; Elias, Joshua E. Cell, Vol. 143, Issue 7 https://doi.org/10.1016/j.cell.2010.12.001	journal	December 2010
Update on designing and building minimal cells Jewett, Michael C.; Forster, Anthony C. Current Opinion in Biotechnology, Vol. 21, Issue 5 https://doi.org/10.1016/j.copbio.2010.06.008	journal	October 2010
Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system Kazuta, Yasuaki; Matsuura, Tomoaki; Ichihashi, Norikazu Journal of Bioscience and Bioengineering, Vol. 118, Issue 5 https://doi.org/10.1016/j.jbiosc.2014.04.019	journal	November 2014
Deconstructing ribosome construction Connolly, Keith; Culver, Gloria Trends in Biochemical Sciences, Vol. 34, Issue 5 https://doi.org/10.1016/j.tibs.2009.01.011	journal	May 2009
Protein synthesis by pure translation systems Shimizu, Yoshihiro; Kanamori, Takashi; Ueda, Takuya Methods, Vol. 36, Issue 3 https://doi.org/10.1016/j.ymeth.2005.04.006	journal	July 2005
The Impact of Peptide Abundance and Dynamic Range on Stable-Isotope-Based Quantitative Proteomic Analyses Bakalarski, Corey E.; Elias, Joshua E.; Villén, Judit Journal of Proteome Research, Vol. 7, Issue 11 https://doi.org/10.1021/pr800333e	journal	September 2008
Multiplexed in Vivo His-Tagging of Enzyme Pathways for in Vitro Single-Pot Multienzyme Catalysis Wang, Harris H.; Huang, Po-Yi; Xu, George ACS Synthetic Biology, Vol. 1, Issue 2, p. 43-52 https://doi.org/10.1021/sb3000029	journal	November 2011
Synthesizing life Szostak, Jack W.; Bartel, David P.; Luisi, P. Luigi Nature, Vol. 409, Issue 6818 https://doi.org/10.1038/35053176	journal	January 2001
Cell-free translation reconstituted with purified components Shimizu, Yoshihiro; Inoue, Akio; Tomari, Yukihide Nature Biotechnology, Vol. 19, Issue 8 https://doi.org/10.1038/90802	journal	August 2001
Proteome‐wide systems analysis of a cellulosic biofuel‐producing microbe Tolonen, Andrew C.; Haas, Wilhelm; Chilaka, Amanda C. Molecular Systems Biology, Vol. 7, Issue 1 https://doi.org/10.1038/msb.2010.116	journal	January 2011
In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation Jewett, Michael C.; Fritz, Brian R.; Timmerman, Laura E. Molecular Systems Biology, Vol. 9, Issue 1 https://doi.org/10.1038/msb.2013.31	journal	January 2013
Refactoring bacteriophage T7 Chan, Leon Y.; Kosuri, Sriram; Endy, Drew Molecular Systems Biology, Vol. 1, Issue 1 https://doi.org/10.1038/msb4100025	journal	January 2005
Towards synthesis of a minimal cell Forster, Anthony C.; Church, George M. Molecular Systems Biology, Vol. 2, Issue 1 https://doi.org/10.1038/msb4100090	journal	January 2006
Accurate multiplex gene synthesis from programmable DNA microchips Tian, Jingdong; Gong, Hui; Sheng, Nijing Nature, Vol. 432, Issue 7020, p. 1050-1054 https://doi.org/10.1038/nature03151	journal	December 2004
Template-directed synthesis of a genetic polymer in a model protocell Mansy, Sheref S.; Schrum, Jason P.; Krishnamurthy, Mathangi Nature, Vol. 454, Issue 7200 https://doi.org/10.1038/nature07018	journal	June 2008
Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry Elias, Joshua E.; Gygi, Steven P. Nature Methods, Vol. 4, Issue 3 https://doi.org/10.1038/nmeth1019	journal	February 2007
Protein synthesis yield increased 72 times in the cell-free PURE system Jackson, Kirsten; Kanamori, Takashi; Ueda, Takuya Integrative Biology, Vol. 6, Issue 8 https://doi.org/10.1039/C4IB00088A	journal	January 2014
RNA, but not protein partners, is directly responsible for translational silencing by a bacterial Hfq-binding small RNA Maki, K.; Uno, K.; Morita, T. Proceedings of the National Academy of Sciences, Vol. 105, Issue 30 https://doi.org/10.1073/pnas.0803106105	journal	July 2008
Generating a synthetic genome by whole genome assembly: φX174 bacteriophage from synthetic oligonucleotides Smith, H. O.; Hutchison, C. A.; Pfannkoch, C. Proceedings of the National Academy of Sciences, Vol. 100, Issue 26, p. 15440-15445 https://doi.org/10.1073/pnas.2237126100	journal	December 2003
Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics Ong, Shao-En; Blagoev, Blagoy; Kratchmarova, Irina Molecular & Cellular Proteomics, Vol. 1, Issue 5 https://doi.org/10.1074/mcp.M200025-MCP200	journal	May 2002
Optimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics Haas, Wilhelm; Faherty, Brendan K.; Gerber, Scott A. Molecular & Cellular Proteomics, Vol. 5, Issue 7 https://doi.org/10.1074/mcp.M500339-MCP200	journal	April 2006
In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme Asahara, Haruichi; Chong, Shaorong Nucleic Acids Research, Vol. 38, Issue 13 https://doi.org/10.1093/nar/gkq377	journal	May 2010
Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity Shabalina, S. A.; Spiridonov, N. A.; Kashina, A. Nucleic Acids Research, Vol. 41, Issue 4 https://doi.org/10.1093/nar/gks1205	journal	January 2013
Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system Li, Jun; Zhang, Chi; Huang, Poyi Translation https://doi.org/10.1101/099838	posted_content	January 2017
The Origins of Cellular Life Schrum, J. P.; Zhu, T. F.; Szostak, J. W. Cold Spring Harbor Perspectives in Biology, Vol. 2, Issue 9 https://doi.org/10.1101/cshperspect.a002212	journal	May 2010
Reconstructing the Emergence of Cellular Life through the Synthesis of Model Protocells Mansy, S. S.; Szostak, J. W. Cold Spring Harbor Symposia on Quantitative Biology, Vol. 74, Issue 0 https://doi.org/10.1101/sqb.2009.74.014	journal	January 2009
The Emergence of Competition Between Model Protocells Chen, I. A. Science, Vol. 305, Issue 5689 https://doi.org/10.1126/science.1100757	journal	September 2004
Emergent Properties of Reduced-Genome Escherichia coli Posfai, G.; Plunkett III, Guy; Fehér, Tamás Science, Vol. 312, Issue 5776, p. 1044-1046 https://doi.org/10.1126/science.1126439	journal	May 2006
Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome Gibson, D. G.; Glass, J. I.; Lartigue, C. Science, Vol. 329, Issue 5987, p. 52-56 https://doi.org/10.1126/science.1190719	journal	May 2010
Improved Cell-Free RNA and Protein Synthesis System Li, Jun; Gu, Liangcai; Aach, John PLoS ONE, Vol. 9, Issue 9 https://doi.org/10.1371/journal.pone.0106232	journal	September 2014
Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples Feist, Peter; Hummon, Amanda International Journal of Molecular Sciences, Vol. 16, Issue 2 https://doi.org/10.3390/ijms16023537	journal	February 2015

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Bacterial cell-free expression technology to in vitro systems engineering and optimization Caschera, Filippo Synthetic and Systems Biotechnology, Vol. 2, Issue 2 https://doi.org/10.1016/j.synbio.2017.07.004	journal	June 2017
In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli Shimojo, Masaru; Amikura, Kazuaki; Masuda, Keiko Communications Biology, Vol. 3, Issue 1 https://doi.org/10.1038/s42003-020-0874-8	journal	March 2020
MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification Takemori, Nobuaki; Takemori, Ayako; Tanaka, Yuki Molecular & Cellular Proteomics, Vol. 16, Issue 12 https://doi.org/10.1074/mcp.ra117.000284	journal	December 2017
Strategies for in vitro engineering of the translation machinery Hammerling, Michael J.; Krüger, Antje; Jewett, Michael C. Nucleic Acids Research, Vol. 48, Issue 3 https://doi.org/10.1093/nar/gkz1011	journal	November 2019
The hallmarks of living systems: towards creating artificial cells Yewdall, N. Amy; Mason, Alexander F.; van Hest, Jan C. M. Interface Focus, Vol. 8, Issue 5 https://doi.org/10.1098/rsfs.2018.0023	journal	August 2018
Gene-Expressing Liposomes as Synthetic Cells for Molecular Communication Studies Rampioni, Giordano; D'Angelo, Francesca; Leoni, Livia Frontiers in Bioengineering and Biotechnology, Vol. 7 https://doi.org/10.3389/fbioe.2019.00001	journal	January 2019
Is Research on “Synthetic Cells” Moving to the Next Level? Stano, Pasquale Life, Vol. 9, Issue 1 https://doi.org/10.3390/life9010003	journal	December 2018
Templated Self‐Replication in Biomimetic Systems Le Vay, Kristian; Weise, Laura Isabel; Libicher, Kai Advanced Biosystems, Vol. 3, Issue 6 https://doi.org/10.1002/adbi.201800313	journal	April 2019
Towards Design of Self‐Organizing Biomimetic Systems Wölfer, Christian; Mangold, Michael; Flassig, Robert J. Advanced Biosystems, Vol. 3, Issue 6 https://doi.org/10.1002/adbi.201800320	journal	March 2019
Mirror‐Image 5S Ribonucleoprotein Complexes Ling, Jun‐Jie; Fan, Chuyao; Qin, Hong Angewandte Chemie, Vol. 132, Issue 9 https://doi.org/10.1002/ange.201914799	journal	January 2020
Mirror‐Image 5S Ribonucleoprotein Complexes Ling, Jun‐Jie; Fan, Chuyao; Qin, Hong Angewandte Chemie International Edition, Vol. 59, Issue 9 https://doi.org/10.1002/anie.201914799	journal	January 2020
Gene Expression Inside Liposomes: From Early Studies to Current Protocols Stano, Pasquale Chemistry – A European Journal, Vol. 25, Issue 33 https://doi.org/10.1002/chem.201806445	journal	April 2019
Regulation of spatiotemporal patterning in artificial cells by a defined protein expression system Yoshida, Aoi; Kohyama, Shunshi; Fujiwara, Kei Chemical Science, Vol. 10, Issue 48 https://doi.org/10.1039/c9sc02441g	journal	January 2019
Reconstitution of 30S ribosomal subunits in vitro using ribosome biogenesis factors Tamaru, Daichi; Amikura, Kazuaki; Shimizu, Yoshihiro RNA, Vol. 24, Issue 11 https://doi.org/10.1261/rna.065615.118	journal	August 2018

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60 APPLIED LIFE SCIENCES
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