Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides

A fundamental goal in biotechnology and biology is the development of approaches to better understand the genetic basis of traits. Here we report a versatile method, trackable multiplex recombineering (TRMR), whereby thousands of specific genetic modifications are created and evaluated simultaneously. To demonstrate TRMR, in a single day we modified the expression of >95% of the genes in Escherichia coli by inserting synthetic DNA cassettes and molecular barcodes upstream of each gene. Barcode sequences and microarrays were then used to quantify population dynamics. Within a week we mapped thousands of genes that affect E. coli growth in various media (rich, minimal and cellulosic hydrolysate) and in the presence of several growth inhibitors (β-glucoside, D-fucose, valine and methylglyoxal). This approach can be applied to a broad range of traits to identify targets for future genome-engineering endeavors.

[1]  K. Murphy,et al.  Use of Bacteriophage λ Recombination Functions To Promote Gene Replacement in Escherichia coli , 1998, Journal of bacteriology.

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

[3]  Corey Nislow,et al.  A unique and universal molecular barcode array , 2006, Nature Methods.

[4]  N. Costantino,et al.  A set of recombineering plasmids for gram-negative bacteria. , 2006, Gene.

[5]  Kia Peyvan,et al.  CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection. , 2007, Biosensors & bioelectronics.

[6]  Farren J. Isaacs,et al.  Programming cells by multiplex genome engineering and accelerated evolution , 2009, Nature.

[7]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S. P. Fodor,et al.  Light-directed, spatially addressable parallel chemical synthesis. , 1991, Science.

[9]  Gilbert Shama,et al.  Estimating the maximum growth rate from microbial growth curves: definition is everything , 2005 .

[10]  A. Klibanov,et al.  Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Michael Zuker,et al.  DINAMelt web server for nucleic acid melting prediction , 2005, Nucleic Acids Res..

[12]  J. D. de Winde,et al.  Engineering Pseudomonas putida S12 for Efficient Utilization of d-Xylose and l-Arabinose , 2008, Applied and Environmental Microbiology.

[13]  Xiaomei Zhou,et al.  Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages , 2008, Proceedings of the National Academy of Sciences.

[14]  Patrick J. Paddison,et al.  Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis , 2004, Nature Methods.

[15]  R. Mowery,et al.  High-performance liquid chromatography method for simultaneous determination of aliphatic acid, aromatic acid and neutral degradation products in biomass pretreatment hydrolysates. , 2006, Journal of chromatography. A.

[16]  F. Dean,et al.  Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. , 2001, Genome research.

[17]  O. Ozier-Kalogeropoulos,et al.  A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. , 1993, Nucleic acids research.

[18]  Corey Nislow,et al.  Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures , 2007, Nature Protocols.

[19]  J. Imlay,et al.  Alkyl Hydroperoxide Reductase Is the Primary Scavenger of Endogenous Hydrogen Peroxide in Escherichia coli , 2001, Journal of bacteriology.

[20]  L. Poole,et al.  Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein reductases. , 2005, Archives of biochemistry and biophysics.

[21]  Overproduction of noncanonical amino acids by Escherichia coli cells , 2007, Microbiology.

[22]  Ryan T Gill,et al.  SCALEs: multiscale analysis of library enrichment , 2007, Nature Methods.

[23]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[24]  L Wodicka,et al.  Parallel analysis of genetic selections using whole genome oligonucleotide arrays. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  C. Sella,et al.  Properties of subcloned subunits of bacterial acetohydroxy acid synthases , 1992, Journal of bacteriology.

[26]  Ronald W. Davis,et al.  Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar–coding strategy , 1996, Nature Genetics.

[27]  James J. Collins,et al.  Next-Generation Synthetic Gene Networks , 2009, Nature Biotechnology.

[28]  D. C. Cameron,et al.  Accumulation of methylglyoxal in anaerobically grown Escherichia coli and its detoxification by expression of the Pseudomonas putida glyoxalase I gene. , 2001, Metabolic engineering.

[29]  J. Collins,et al.  A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics , 2007, Cell.

[30]  D. Schell,et al.  Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol. , 2009, Biotechnology and bioengineering.

[31]  J. Imlay,et al.  The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli , 1999, Molecular microbiology.

[32]  M. Sussman,et al.  Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array , 1999, Nature Biotechnology.

[33]  Ronald W. Davis,et al.  Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. , 1999, Science.

[34]  G. Stephanopoulos,et al.  Genome-wide screening for trait conferring genes using DNA microarrays , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[35]  John C Whitman,et al.  Improving catalytic function by ProSAR-driven enzyme evolution , 2007, Nature Biotechnology.

[36]  J. Shendure,et al.  Selection analyses of insertional mutants using subgenic-resolution arrays , 2001, Nature Biotechnology.

[37]  D. Court,et al.  High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[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]  Morten H. H. Nørholm,et al.  Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments , 2006, Nucleic acids research.

[40]  M. Kimura,et al.  Blasticidin S deaminase gene from Aspergillus terreus (BSD): a new drug resistance gene for transfection of mammalian cells. , 1994, Biochimica et biophysica acta.

[41]  H. Mori,et al.  Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. , 2006, DNA research : an international journal for rapid publication of reports on genes and genomes.

[42]  J. Shine,et al.  The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[43]  A. Blanchard,et al.  High-density oligonucleotide arrays , 1996 .

[44]  Ronald W. Davis,et al.  Functional profiling of the Saccharomyces cerevisiae genome , 2002, Nature.

[45]  D. Court,et al.  An efficient recombination system for chromosome engineering in Escherichia coli. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Nicholas J Turner,et al.  Directed evolution drives the next generation of biocatalysts. , 2009, Nature chemical biology.

[47]  D. Botstein,et al.  A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds , 2009, Nature Biotechnology.

[48]  S. Wessler,et al.  Control of leu operon expression in Escherichia coli by a transcription attenuation mechanism. , 1981, Journal of molecular biology.

[49]  M. De Felice,et al.  Cryptic operon for beta-glucoside metabolism in Escherichia coli K12: genetic evidence for a regulatory protein. , 1981, Genetics.