Multi-Input Regulation and Logic with T7 Promoters in Cells and Cell-Free Systems

Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on native host promoters for the construction of circuit elements, such as logic gates, can make the implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell-free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell-free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in an E. coli cell-free protein expression system. Our results expand the utility of T7 promoters in cell based as well as cell-free synthetic biology applications.

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

[2]  W. Mcallister.  Transcription by T7 RNA Polymerase , 1997 .

[3]  Christopher A. Voigt,et al.  A Synthetic Genetic Edge Detection Program , 2009, Cell.

[4]  S. Retterer,et al.  Enzyme reactions in nanoporous, picoliter volume containers. , 2012, Analytical chemistry.

[5]  Kate Thodey,et al.  Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways. , 2012, Metabolic engineering.

[6]  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.

[7]  B. Müller-Hill,et al.  Four dimers of lambda repressor bound to two suitably spaced pairs of lambda operators form octamers and DNA loops over large distances. , 1999, Current biology : CB.

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

[9]  J. Collins,et al.  Synthetic Biology Moving into the Clinic , 2011, Science.

[10]  E. Andrianantoandro,et al.  Synthetic biology: new engineering rules for an emerging discipline , 2006, Molecular systems biology.

[11]  Hernan G. Garcia,et al.  Concentration and Length Dependence of DNA Looping in Transcriptional Regulation , 2008, PLoS ONE.

[12]  Gregory S. Snider,et al.  ‘Memristive’ switches enable ‘stateful’ logic operations via material implication , 2010, Nature.

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

[14]  B. Müller-Hill,et al.  Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator. , 1996, Journal of molecular biology.

[15]  R. R. Burgess,et al.  Construction of bacteriophage T7 late promoters with point mutations and characterization by in vitro transcription properties , 1987, Nucleic Acids Res..

[16]  L. J. Maher,et al.  Inhibition of T7 RNA polymerase initiation by triple-helical DNA complexes: a model for artificial gene repression. , 1992, Biochemistry.

[17]  Ahmad S. Khalil,et al.  A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions , 2012, Cell.

[18]  C. Richardson,et al.  A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Drew Endy,et al.  Amplifying Genetic Logic Gates , 2013, Science.

[20]  Jian Zhan,et al.  Develop reusable and combinable designs for transcriptional logic gates , 2010, Molecular systems biology.

[21]  Christopher A. Voigt,et al.  Environmental signal integration by a modular AND gate , 2007, Molecular systems biology.

[22]  M. Isalan,et al.  Engineering Gene Networks to Emulate Drosophila Embryonic Pattern Formation , 2005, PLoS biology.

[23]  M. Elowitz,et al.  A synthetic oscillatory network of transcriptional regulators , 2000, Nature.

[24]  M. L. Simpson,et al.  Nano-enabled synthetic biology , 2007, Molecular systems biology.

[25]  M. Dreyfus,et al.  On the mechanism of inhibition of phage T7 RNA polymerase by lac repressor. , 1998, Journal of molecular biology.

[26]  J. Chin,et al.  Synthesis of orthogonal transcription-translation networks , 2009, Proceedings of the National Academy of Sciences.

[27]  F. Studier,et al.  Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. , 1991, Journal of molecular biology.

[28]  Benno Müller-Hill,et al.  Repression oflacPromoter as a Function of Distance, Phase and Quality of an AuxiliarylacOperator , 1996 .

[29]  S. Adhya,et al.  Control of transcription of gal repressor and isorepressor genes in Escherichia coli , 1993, Journal of bacteriology.

[30]  Ilias Tagkopoulos,et al.  A synthetic biology approach to self-regulatory recombinant protein production in Escherichia coli , 2012, Journal of biological engineering.

[31]  B. Müller-Hill,et al.  The three operators of the lac operon cooperate in repression. , 1990, The EMBO journal.

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

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

[34]  M. L. Simpson,et al.  Micro/nanofabricated environments for synthetic biology. , 2011, Current opinion in biotechnology.

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

[36]  C. Richardson,et al.  A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. 1985. , 1992, Biotechnology.

[37]  Zhen Xie,et al.  Logic integration of mRNA signals by an RNAi-based molecular computer , 2010, Nucleic acids research.

[38]  D. Hensley,et al.  Microarrays of Biomimetic Cells Formed by the Controlled Synthesis of Carbon Nanofiber Membranes , 2004 .

[39]  Peter G Schultz,et al.  An Expanded Eukaryotic Genetic Code , 2003, Science.

[40]  Tetsuya Yomo,et al.  Expression of a cascading genetic network within liposomes , 2004, FEBS letters.

[41]  R. Bar-Ziv,et al.  Cell-free protein synthesis and assembly on a biochip. , 2012, Nature nanotechnology.

[42]  Benno Müller-Hill,et al.  Four dimers of λ repressor bound to two suitably spaced pairs of λ operators form octamers and DNA loops over large distances , 1999, Current Biology.

[43]  S. Busby,et al.  The regulation of bacterial transcription initiation , 2004, Nature Reviews Microbiology.

[44]  Joy Sinha,et al.  Reprogramming Bacteria to Seek and Destroy a Herbicide , 2010, Nature chemical biology.

[45]  Michael C Jewett,et al.  Synthetic in vitro circuits. , 2012, Current opinion in chemical biology.

[46]  Christopher A. Voigt,et al.  Genetic programs constructed from layered logic gates in single cells , 2012, Nature.

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

[48]  C. V. Rao,et al.  Expanding the synthetic biology toolbox: engineering orthogonal regulators of gene expression. , 2012, Current opinion in biotechnology.

[49]  L. You,et al.  Emergent bistability by a growth-modulating positive feedback circuit. , 2009, Nature chemical biology.

[50]  Thomas H. Segall-Shapiro,et al.  Modular control of multiple pathways using engineered orthogonal T7 polymerases , 2012, Nucleic acids research.

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

[52]  D. Lilley,et al.  Mechanisms of Transcription , 1997, Nucleic Acids and Molecular Biology.

[53]  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.

[54]  Matthew R Bennett,et al.  Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants , 2013, Proceedings of the National Academy of Sciences.

[55]  David K. Karig,et al.  Signal-amplifying genetic circuit enables in vivo observation of weak promoter activation in the Rhl quorum sensing system. , 2005, Biotechnology and bioengineering.

[56]  Richard H. Ebright,et al.  Promoter structure, promoter recognition, and transcription activation in prokaryotes , 1994, Cell.

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

[58]  R. Schleif,et al.  Variation of half‐site organization and DNA looping by AraC protein. , 1993, The EMBO journal.

[59]  R. Schleif,et al.  DNA looping. , 1988, Science.

[60]  W. Mcallister,et al.  T7 RNA polymerase mutants with altered promoter specificities. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[61]  B. Müller-Hill,et al.  Quality and position of the three lac operators of E. coli define efficiency of repression. , 1994, The EMBO journal.

[62]  Christopher A. Voigt,et al.  Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’ , 2011, Nature.

[63]  Timothy S. Ham,et al.  Production of the antimalarial drug precursor artemisinic acid in engineered yeast , 2006, Nature.

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

[65]  Roger Y. Tsien,et al.  Improved green fluorescence , 1995, Nature.

[66]  Elizabeth Guruceaga,et al.  Adenovirus VA RNA-derived miRNAs target cellular genes involved in cell growth, gene expression and DNA repair , 2009, Nucleic acids research.

[67]  J. Kahn,et al.  Gene repression by minimal lac loops in vivo , 2010, Nucleic acids research.

[68]  Griffin M. Weber,et al.  BioNumbers—the database of key numbers in molecular and cell biology , 2009, Nucleic Acids Res..

[69]  Sandeep Krishna,et al.  Genetic flexibility of regulatory networks , 2010, Proceedings of the National Academy of Sciences.