Characterizing and Prototyping Genetic Networks with Cell-Free Transcription-Translation Reactions

A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative ‘design-build-test’ cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription-translation (TX-TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX-TL system that utilizes the native Escherichia coli TX-TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX-TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX-TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems. Abbreviations TX-TL (transcription-translation), I1-FFL (incoherent feed-forward loop type 1) GFP (green fluorescent protein)

[1]  E. Wagner,et al.  Antisense RNA‐mediated transcriptional attenuation: an in vitro study of plasmid pT181 , 2000, Molecular microbiology.

[2]  Richard M. Murray,et al.  A cell-free framework for biological systems engineering , 2015, bioRxiv.

[3]  Alfonso Jaramillo,et al.  RiboMaker: computational design of conformation-based riboregulation , 2014, Bioinform..

[4]  Matthias Heinemann,et al.  Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis. , 2011, Nature chemical biology.

[5]  G Svenneby,et al.  [Enzymatic reaction mechanisms]. , 1970, Tidsskrift for den Norske laegeforening : tidsskrift for praktisk medicin, ny raekke.

[6]  Dong-Myung Kim,et al.  Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. , 2001, Biotechnology and bioengineering.

[7]  A. Spirin,et al.  A continuous cell-free translation system capable of producing polypeptides in high yield. , 1988, Science.

[8]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[9]  Christopher A. Voigt,et al.  Automated design of synthetic ribosome binding sites to control protein expression , 2016 .

[10]  Richard M. Murray,et al.  Biomolecular resource utilization in elementary cell-free gene circuits , 2013, 2013 American Control Conference.

[11]  J. Keasling.  Synthetic biology for synthetic chemistry. , 2008, ACS chemical biology.

[12]  Joeri Beauprez,et al.  One step DNA assembly for combinatorial metabolic engineering. , 2014, Metabolic engineering.

[13]  Takuya Ueda,et al.  Cell-free translation reconstituted with purified components , 2001, Nature Biotechnology.

[14]  Paul S. Freemont,et al.  Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology , 2013, Nucleic acids research.

[15]  Drew Endy,et al.  Precise and reliable gene expression via standard transcription and translation initiation elements , 2013, Nature Methods.

[16]  Talmon Arad,et al.  Cell-free co-synthesis of protein nanoassemblies: tubes, rings, and doughnuts. , 2007, Nano letters.

[17]  T. Terwilliger,et al.  Engineering and characterization of a superfolder green fluorescent protein , 2006, Nature Biotechnology.

[18]  Uri Alon,et al.  Response delays and the structure of transcription networks. , 2003, Journal of molecular biology.

[19]  David K. Karig,et al.  Expression optimization and synthetic gene networks in cell-free systems , 2011, Nucleic acids research.

[20]  Jim Swartz,et al.  Developing cell-free biology for industrial applications , 2006, Journal of Industrial Microbiology and Biotechnology.

[21]  James R. Swartz,et al.  Site-specific incorporation of p-propargyloxyphenylalanine in a cell-free environment for direct protein-protein click conjugation. , 2010, Bioconjugate chemistry.

[22]  Michael C Jewett.  Cell-free synthetic biology special issue. , 2014, ACS synthetic biology.

[23]  Adam P Arkin,et al.  Versatile RNA-sensing transcriptional regulators for engineering genetic networks , 2011, Proceedings of the National Academy of Sciences.

[24]  Uri Alon,et al.  An Introduction to Systems Biology , 2006 .

[25]  Jung-Won Keum,et al.  Prolonged cell‐free protein synthesis using dual energy sources: Combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate , 2007, Biotechnology and bioengineering.

[26]  R. Bar-Ziv,et al.  Protein nanomachines assembly modes: cell-free expression and biochip perspectives. , 2013, Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology.

[27]  Priscilla E. M. Purnick,et al.  The second wave of synthetic biology: from modules to systems , 2009, Nature Reviews Molecular Cell Biology.

[28]  Kate Thomas.  The Past , 2015 .

[29]  Richard M. Murray,et al.  Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription–Translation (TX-TL) Systems , 2014, ACS synthetic biology.

[30]  H. Bujard,et al.  Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. , 1997, Nucleic acids research.

[31]  Joseph H. Davis,et al.  Design, construction and characterization of a set of insulated bacterial promoters , 2010, Nucleic acids research.

[32]  Dong-Myung Kim,et al.  Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source , 2011 .

[33]  J. Collins,et al.  A brief history of synthetic biology , 2014, Nature Reviews Microbiology.

[34]  Richard M. Murray,et al.  Protein degradation in a TX-TL cell-free expression system using ClpXP protease , 2014, bioRxiv.

[35]  R. Bar-Ziv,et al.  Principles of cell-free genetic circuit assembly , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[36]  V. Noireaux,et al.  An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. , 2012, ACS synthetic biology.

[37]  Rui Gan,et al.  Cell-free protein synthesis: applications come of age. , 2012, Biotechnology advances.

[38]  Michael C Jewett,et al.  An integrated cell-free metabolic platform for protein production and synthetic biology , 2008, Molecular systems biology.

[39]  Dong-Myung Kim,et al.  Prolonging cell-free protein synthesis with a novel ATP regeneration system. , 1999, Biotechnology and bioengineering.

[40]  J. H. Matthaei,et al.  Approximation of genetic code via cell-free protein synthesis directed by template RNA. , 1963, Federation proceedings.

[41]  R. Murray,et al.  Gene circuit performance characterization and resource usage in a cell-free "breadboard". , 2014, ACS synthetic biology.

[42]  Dominic Esposito,et al.  A novel cell-free protein synthesis system. , 2004, Journal of biotechnology.

[43]  Vincent Noireaux,et al.  Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. , 2012, ACS synthetic biology.

[44]  James J. Collins,et al.  Paper-Based Synthetic Gene Networks , 2014, Cell.

[45]  J. Ahn,et al.  Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions. , 2005, Biochemical and biophysical research communications.

[46]  Tom Ellis,et al.  Modelling the burden caused by gene expression: an in silico investigation into the interactions between synthetic gene circuits and their chassis cell , 2013, 1309.7798.

[47]  Henrike Niederholtmeyer,et al.  Implementation of cell-free biological networks at steady state , 2013, Proceedings of the National Academy of Sciences.

[48]  G. Church,et al.  Large-scale de novo DNA synthesis: technologies and applications , 2014, Nature Methods.

[49]  James Chappell,et al.  Creating small transcription activating RNAs. , 2015, Nature chemical biology.

[50]  Julius B. Lucks,et al.  Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals , 2012, Nucleic acids research.

[51]  Malra C. Treece.  An A is an A is an A is an E. , 1976 .

[52]  James Swartz,et al.  Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome. , 2004, Metabolic engineering.

[53]  J. Kornblum,et al.  pT181 plasmid replication is regulated by a countertranscript-driven transcriptional attenuator , 1989, Cell.

[54]  Bradley Charles Bundy,et al.  Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates. , 2014, New biotechnology.

[55]  Vincent Noireaux,et al.  Coarse-grained dynamics of protein synthesis in a cell-free system. , 2011, Physical review letters.

[56]  Ahmad S. Khalil,et al.  Synthetic biology: applications come of age , 2010, Nature Reviews Genetics.

[57]  Richard M. Murray,et al.  Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology , 2013, Journal of visualized experiments : JoVE.

[58]  Vincent Noireaux,et al.  Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70 , 2010, Journal of biological engineering.

[59]  J. Collins,et al.  Toehold Switches: De-Novo-Designed Regulators of Gene Expression , 2014, Cell.

[60]  Douglas Densmore,et al.  Interactive assembly algorithms for molecular cloning , 2014, Nature Methods.

[61]  Jay D Keasling,et al.  Model-Driven Engineering of RNA Devices to Quantitatively Program Gene Expression , 2011, Science.

[62]  Tania A. Baker,et al.  Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines , 2005, Nature.

[63]  Vincent Noireaux,et al.  Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system. , 2014, ACS synthetic biology.

[64]  Vincent Noireaux,et al.  Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system , 2010, Journal of biological engineering.

[65]  Ernst Weber,et al.  A Modular Cloning System for Standardized Assembly of Multigene Constructs , 2011, PloS one.

[66]  Mingyue He,et al.  Cell-free protein synthesis: applications in proteomics and biotechnology. , 2008, New biotechnology.

[67]  Carola Engler,et al.  A One Pot, One Step, Precision Cloning Method with High Throughput Capability , 2008, PloS one.

[68]  R. Weiss,et al.  Cancer Cells Multi-Input RNAi-Based Logic Circuit for Identification of Specific , 2011 .

[69]  Domitilla Del Vecchio,et al.  Limitations and trade-offs in gene expression due to competition for shared cellular resources , 2014, CDC.

[70]  Andrew D Griffiths,et al.  A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution. , 2012, Lab on a chip.

[71]  J. Swartz,et al.  Cell‐free synthesis of proteins that require disulfide bonds using glucose as an energy source , 2007, Biotechnology and bioengineering.

[72]  T. Baker,et al.  Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. , 2003, Molecular cell.

[73]  Michael C. Jewett,et al.  Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis , 2014, Front. Chem..

[74]  Y Cenatiempo,et al.  Prokaryotic gene expression in vitro: transcription-translation coupled systems. , 1986, Biochimie.

[75]  Vincent Noireaux,et al.  Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. , 2014, Biochimie.

[76]  Henrike Niederholtmeyer,et al.  Real-time mRNA measurement during an in vitro transcription and translation reaction using binary probes. , 2013, ACS synthetic biology.

[77]  S. Mangan,et al.  Structure and function of the feed-forward loop network motif , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[78]  M. Chamberlin,et al.  Secondary sigma factor controls transcription of flagellar and chemotaxis genes in Escherichia coli. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[79]  Ashty S. Karim,et al.  Cell‐free metabolic engineering: Biomanufacturing beyond the cell , 2015, Biotechnology journal.

[80]  James R. Swartz,et al.  High‐level cell‐free synthesis yields of proteins containing site‐specific non‐natural amino acids , 2009, Biotechnology and bioengineering.

[81]  Jung-Won Keum,et al.  Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system. , 2006, Journal of biotechnology.

[82]  Gerry White,et al.  The Past , 2000 .

[83]  Christian R. Boehm,et al.  Unique nucleotide sequence–guided assembly of repetitive DNA parts for synthetic biology applications , 2014, Nature Protocols.

[84]  Y.‐H.P. Zhang,et al.  Cell-free protein synthesis energized by slowly-metabolized maltodextrin , 2009, BMC biotechnology.

[85]  Geoffrey Chang,et al.  The past, present and future of cell-free protein synthesis. , 2005, Trends in biotechnology.

[86]  Marshall Nirenberg,et al.  Historical review: Deciphering the genetic code--a personal account. , 2004, Trends in biochemical sciences.

[87]  J. Swartz,et al.  Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell‐Free Protein Synthesis , 2008, Biotechnology progress.

[88]  S. Zimmerman,et al.  Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. , 1991, Journal of molecular biology.

[89]  C. J. Murray,et al.  Microscale to Manufacturing Scale-up of Cell-Free Cytokine Production—A New Approach for Shortening Protein Production Development Timelines , 2011, Biotechnology and bioengineering.

[90]  Yasuhiko Yoshida,et al.  Cell‐free production and stable‐isotope labeling of milligram quantities of proteins , 1999, FEBS letters.

[91]  Michael C. Jewett,et al.  High-throughput preparation methods of crude extract for robust cell-free protein synthesis , 2015, Scientific Reports.

[92]  J. Keasling,et al.  Integrating Biological Redesign: Where Synthetic Biology Came From and Where It Needs to Go , 2014, Cell.

[93]  Sriram Kosuri,et al.  Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips , 2010, Nature Biotechnology.

[94]  Jonathan A. Goler,et al.  Chemical synthesis using synthetic biology. , 2009, Current opinion in biotechnology.

[95]  P ? ? ? ? ? ? ? % ? ? ? ? , 1991 .

[96]  P. Stadler,et al.  De novo design of a synthetic riboswitch that regulates transcription termination , 2012, Nucleic acids research.

[97]  Kei Fujiwara,et al.  Condensation of an Additive-Free Cell Extract to Mimic the Conditions of Live Cells , 2013, PloS one.

[98]  Vincent Noireaux,et al.  Programmable on-chip DNA compartments as artificial cells , 2014, Science.

[99]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[100]  Jeffrey D Varner,et al.  Generating Effective Models and Parameters for RNA Genetic Circuits. , 2015, ACS synthetic biology.

[101]  Jim Euchner.  Design , 2014, Catalysis from A to Z.

[102]  Bradley Charles Bundy,et al.  Streamlined extract preparation for Escherichia coli-based cell-free protein synthesis by sonication or bead vortex mixing. , 2012, BioTechniques.

[103]  Vincent Noireaux,et al.  A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system. , 2015, Metabolic engineering.

[104]  James Swartz,et al.  Increasing PCR Fragment Stability and Protein Yields in a Cell-Free System with Genetically Modified Escherichia coli Extracts , 2005, Journal of Molecular Microbiology and Biotechnology.