Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

[1]  David K. Karig,et al.  Noise in biological circuits. , 2009, Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology.

[2]  Vincent Noireaux,et al.  A vesicle bioreactor as a step toward an artificial cell assembly. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[3]  C. Collier,et al.  Single-molecule mobility in confined and crowded femtolitre chambers. , 2013, Lab on a chip.

[4]  C. Rao,et al.  Control, exploitation and tolerance of intracellular noise , 2002, Nature.

[5]  G. Blobel,et al.  Cohesin subunit SMC1 associates with mitotic microtubules at the spindle pole , 2008, Proceedings of the National Academy of Sciences.

[6]  Chris D Cox,et al.  Using noise to probe and characterize gene circuits , 2008, Proceedings of the National Academy of Sciences.

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

[8]  Jakob Stoustrup,et al.  Direct control implementation of a refrigeration system in smart grid , 2013, 2013 American Control Conference.

[9]  N. Stanietsky,et al.  The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity , 2009, Proceedings of the National Academy of Sciences.

[10]  A. van Oudenaarden,et al.  Noise Propagation in Gene Networks , 2005, Science.

[11]  T. Elston,et al.  Stochasticity in gene expression: from theories to phenotypes , 2005, Nature Reviews Genetics.

[12]  Avraham Rasooly,et al.  Biomolecular separation and analysis , 2009 .

[13]  A. Piruska,et al.  Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate , 2013, Proceedings of the National Academy of Sciences.

[14]  S. Quake,et al.  Monolithic microfabricated valves and pumps by multilayer soft lithography. , 2000, Science.

[15]  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.

[16]  Heinz Rüterjans,et al.  High level cell-free expression and specific labeling of integral membrane proteins. , 2004, European journal of biochemistry.

[17]  M. L. Simpson,et al.  Gene network shaping of inherent noise spectra , 2006, Nature.

[18]  P Sarnow,et al.  Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae , 1994, Molecular and cellular biology.

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

[20]  M. Jewett,et al.  Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm , 2004, Biotechnology and bioengineering.

[21]  Mads Kærn,et al.  Noise in eukaryotic gene expression , 2003, Nature.

[22]  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.

[23]  K. Yoshikawa,et al.  Cell-Sized confinement in microspheres accelerates the reaction of gene expression , 2012, Scientific Reports.

[24]  James C. W. Locke,et al.  Using movies to analyse gene circuit dynamics in single cells , 2009, Nature Reviews Microbiology.

[25]  Rajan P Kulkarni,et al.  Tunability and Noise Dependence in Differentiation Dynamics , 2007, Science.

[26]  Stergios Logothetidis,et al.  Nanomedicine and Nanobiotechnology , 2012 .

[27]  Kenichi Yoshikawa,et al.  Gene Expression within Cell‐Sized Lipid Vesicles , 2003, Chembiochem : a European journal of chemical biology.

[28]  M. L. Simpson,et al.  Transient-mediated fate determination in a transcriptional circuit of HIV , 2007, Nature Genetics.

[29]  Pasquale Stano,et al.  The Minimal Size of Liposome‐Based Model Cells Brings about a Remarkably Enhanced Entrapment and Protein Synthesis , 2009, Chembiochem : a European journal of chemical biology.

[30]  R. Segev,et al.  GENERAL PROPERTIES OF THE TRANSCRIPTIONAL TIME-SERIES IN ESCHERICHIA COLI , 2011, Nature Genetics.

[31]  M. L. Simpson,et al.  Whole-cell biocomputing. , 2001, Trends in biotechnology.

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

[33]  M. L. Simpson,et al.  Frequency domain analysis of noise in autoregulated gene circuits , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Mikkel A. Algire,et al.  Development and characterization of a reconstituted yeast translation initiation system. , 2002, RNA.

[35]  Michael L. Simpson,et al.  Transcriptional burst frequency and burst size are equally modulated across the human genome , 2012, Proceedings of the National Academy of Sciences.

[36]  Cheemeng Tan,et al.  Molecular crowding shapes gene expression in synthetic cellular nanosystems , 2013, Nature nanotechnology.

[37]  Piro Siuti,et al.  Continuous protein production in nanoporous, picolitre volume containers. , 2011, Lab on a chip.

[38]  A. Arkin,et al.  Stochastic mechanisms in gene expression. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[39]  A. Arkin,et al.  Stochastic kinetic analysis of developmental pathway bifurcation in phage lambda-infected Escherichia coli cells. , 1998, Genetics.

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

[41]  Jeffrey W. Smith,et al.  Stochastic Gene Expression in a Single Cell , 2022 .

[42]  Dirk van Swaay,et al.  Microfluidic methods for forming liposomes. , 2013, Lab on a chip.

[43]  Christophe Danelon,et al.  Linking genotype and phenotype in protein synthesizing liposomes with external supply of resources. , 2013, ACS synthetic biology.

[44]  S. Retterer,et al.  Development and fabrication of nanoporous silicon-based bioreactors within a microfluidic chip. , 2010, Lab on a chip.

[45]  Shinji Okazaki,et al.  Pushing the limits of lithography , 2000, Nature.

[46]  J. Vilar,et al.  From molecular noise to behavioural variability in a single bacterium , 2004, Nature.

[47]  Tetsuya Yomo,et al.  Cell-free protein synthesis from a single copy of DNA in a glass microchamber. , 2012, Lab on a chip.

[48]  M. L. Simpson,et al.  Probing cell-free gene expression noise in femtoliter volumes. , 2013, ACS synthetic biology.

[49]  Soong Ho Um,et al.  A cell-free protein-producing gel. , 2009, Nature materials.

[50]  A. Oudenaarden,et al.  Nature, Nurture, or Chance: Stochastic Gene Expression and Its Consequences , 2008, Cell.

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

[52]  Jared E. Toettcher,et al.  Stochastic Gene Expression in a Lentiviral Positive-Feedback Loop: HIV-1 Tat Fluctuations Drive Phenotypic Diversity , 2005, Cell.