Quantitative estimation of activity and quality for collections of functional genetic elements

The practice of engineering biology now depends on the ad hoc reuse of genetic elements whose precise activities vary across changing contexts. Methods are lacking for researchers to affordably coordinate the quantification and analysis of part performance across varied environments, as needed to identify, evaluate and improve problematic part types. We developed an easy-to-use analysis of variance (ANOVA) framework for quantifying the performance of genetic elements. For proof of concept, we assembled and analyzed combinations of prokaryotic transcription and translation initiation elements in Escherichia coli. We determined how estimation of part activity relates to the number of unique element combinations tested, and we show how to estimate expected ensemble-wide part activity from just one or two measurements. We propose a new statistic, biomolecular part 'quality', for tracking quantitative variation in part performance across changing contexts.

[1]  Christina D Smolke,et al.  Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems , 2010, Proceedings of the National Academy of Sciences.

[2]  T. D. Schneider,et al.  Quantitative analysis of ribosome binding sites in E.coli. , 1994, Nucleic acids research.

[3]  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.

[4]  Gabriel C. Wu,et al.  Successes and failures in modular genetic engineering. , 2012, Current Opinion in Chemical Biology.

[5]  India G. Hook-Barnard,et al.  Transcription Initiation by Mix and Match Elements: Flexibility for Polymerase Binding to Bacterial Promoters , 2007, Gene regulation and systems biology.

[6]  Christopher A. Voigt,et al.  Environmentally controlled invasion of cancer cells by engineered bacteria. , 2006, Journal of molecular biology.

[7]  M. Chamberlin,et al.  Sequences linked to prokaryotic promoters can affect the efficiency of downstream termination sites. , 1989, Journal of molecular biology.

[8]  Adam P Arkin,et al.  Toward rational design of bacterial genomes. , 2011, Current opinion in microbiology.

[9]  D. Endy,et al.  Refinement and standardization of synthetic biological parts and devices , 2008, Nature Biotechnology.

[10]  G. Churchill,et al.  Experimental design for gene expression microarrays. , 2001, Biostatistics.

[11]  J. Keasling Manufacturing Molecules Through Metabolic Engineering , 2010, Science.

[12]  Jason Micklefield,et al.  Mining and engineering natural-product biosynthetic pathways. , 2007, Nature chemical biology.

[13]  T. Funatsu,et al.  Kinetic study of de novo chromophore maturation of fluorescent proteins. , 2011, Analytical biochemistry.

[14]  K. Shearwin,et al.  Transcriptional interference--a crash course. , 2005, Trends in genetics : TIG.

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

[16]  G. Stormo,et al.  Translation initiation in Escherichia coli: sequences within the ribosome‐binding site , 1992, Molecular microbiology.

[17]  Chueh Loo Poh,et al.  Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen , 2011, Molecular systems biology.

[18]  J. Keasling,et al.  Library of Synthetic 5′ Secondary Structures To Manipulate mRNA Stability in Escherichia coli , 1999, Biotechnology progress.

[19]  G. Stephanopoulos,et al.  Tuning genetic control through promoter engineering. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Steven E. Lindow,et al.  Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria , 2001, Journal of bacteriology.

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

[22]  Raphael Gottardo,et al.  flowClust: a Bioconductor package for automated gating of flow cytometry data , 2009, BMC Bioinformatics.

[23]  M. Chamberlin,et al.  Parameters affecting transcription termination by Escherichia coli RNA. II. Construction and analysis of hybrid terminators. , 1992, Journal of molecular biology.

[24]  Gary A. Churchill,et al.  Analysis of Variance for Gene Expression Microarray Data , 2000, J. Comput. Biol..

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

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

[27]  H. Bujard,et al.  Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. , 1982, The EMBO journal.

[28]  Meghdad Hajimorad,et al.  BglBrick vectors and datasheets: A synthetic biology platform for gene expression , 2011, Journal of biological engineering.

[29]  Nathan J Hillson,et al.  j5 DNA assembly design automation software. , 2012, ACS synthetic biology.

[30]  D. Endy,et al.  Rewritable digital data storage in live cells via engineered control of recombination directionality , 2012, Proceedings of the National Academy of Sciences.

[31]  Erik Remaut,et al.  Tight Transcriptional Control Mechanism Ensures Stable High-Level Expression from T7 Promoter-Based Expression Plasmids , 1995, Bio/Technology.

[32]  H. Bujard,et al.  Context-dependent effects of upstream A-tracts. Stimulation or inhibition of Escherichia coli promoter function. , 1994, Journal of molecular biology.

[33]  Adam P Arkin,et al.  Supplementary information for Rationally designed families of orthogonal RNA regulators of translation , 2012 .

[34]  P. V. von Hippel,et al.  A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli. , 1991, Biochemistry.

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

[36]  D. Endy Foundations for engineering biology , 2005, Nature.

[37]  A. Ishihama,et al.  Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector , 2004, Journal of bacteriology.

[38]  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.

[39]  S Wold,et al.  Quantitative sequence-activity models (QSAM)--tools for sequence design. , 1993, Nucleic acids research.

[40]  Keith Dudley Short protocols in molecular biology , 1990 .

[41]  U. Alon,et al.  A comprehensive library of fluorescent transcriptional reporters for Escherichia coli , 2006, Nature Methods.

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

[43]  H. Gulvanessian,et al.  Eurocodes: using reliability analysis to combine action effects , 2005 .

[44]  J. van Duin,et al.  Control of translation by mRNA secondary structure in Escherichia coli. A quantitative analysis of literature data. , 1994, Journal of molecular biology.

[45]  B. Wanner,et al.  Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria , 2001, Journal of bacteriology.

[46]  Christopher A. Voigt,et al.  Ribozyme-based insulator parts buffer synthetic circuits from genetic context , 2012, Nature Biotechnology.

[47]  A. Arkin,et al.  Contextualizing context for synthetic biology – identifying causes of failure of synthetic biological systems , 2012, Biotechnology journal.

[48]  Christina D. Smolke,et al.  Synthetic RNA modules for fine-tuning gene expression levels in yeast by modulating RNase III activity , 2011, Nucleic acids research.

[49]  Peter A Carr,et al.  Genome engineering , 2009, Nature Biotechnology.

[50]  Drew Endy,et al.  Measuring the activity of BioBrick promoters using an in vivo reference standard , 2009, Journal of biological engineering.

[51]  Jeremy Minshull,et al.  Engineering the Salmonella type III secretion system to export spider silk monomers , 2009, Molecular systems biology.

[52]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[53]  T. Hwa,et al.  Growth Rate-Dependent Global Effects on Gene Expression in Bacteria , 2009, Cell.

[54]  Adam P Arkin,et al.  RNA processing enables predictable programming of gene expression , 2012, Nature Biotechnology.

[55]  J. Collins,et al.  DIVERSITY-BASED, MODEL-GUIDED CONSTRUCTION OF SYNTHETIC GENE NETWORKS WITH PREDICTED FUNCTIONS , 2009, Nature Biotechnology.

[56]  Christina D Smolke,et al.  Building outside of the box: iGEM and the BioBricks Foundation , 2009, Nature Biotechnology.

[57]  C. Yanofsky,et al.  Sequence changes preceding a Shine-Dalgarno region influence trpE mRNA translation and decay. , 1988, Journal of molecular biology.

[58]  M. Elowitz,et al.  Programming gene expression with combinatorial promoters , 2007, Molecular systems biology.

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

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

[61]  M. Dreyfus,et al.  Interdependence of translation, transcription and mRNA degradation in the lacZ gene. , 1992, Journal of molecular biology.

[62]  Vivek K. Mutalik,et al.  Promoter Strength Properties of the Complete Sigma E Regulon of Escherichia coli and Salmonella enterica , 2009, Journal of bacteriology.

[63]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.