Registry in a tube: multiplexed pools of retrievable parts for genetic design space exploration

Genetic designs can consist of dozens of genes and hundreds of genetic parts. After evaluating a design, it is desirable to implement changes without the cost and burden of starting the construction process from scratch. Here, we report a two-step process where a large design space is divided into deep pools of composite parts, from which individuals are retrieved and assembled to build a final construct. The pools are built via multiplexed assembly and sequenced using next-generation sequencing. Each pool consists of ∼20 Mb of up to 5000 unique and sequence-verified composite parts that are barcoded for retrieval by PCR. This approach is applied to a 16-gene nitrogen fixation pathway, which is broken into pools containing a total of 55 848 composite parts (71.0 Mb). The pools encompass an enormous design space (1043 possible 23 kb constructs), from which an algorithmguided 192-member 4.5 Mb library is built. Next, all 1030 possible genetic circuits based on 10 repressors (NOR/NOT gates) are encoded in pools where each repressor is fused to all permutations of input promoters. These demonstrate that multiplexing can be applied to encompass entire design spaces from which individuals can be accessed and evaluated.

[1]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.

[2]  Herbert M Sauro,et al.  Randomized BioBrick assembly: a novel DNA assembly method for randomizing and optimizing genetic circuits and metabolic pathways. , 2013, ACS synthetic biology.

[3]  Christopher A. Voigt,et al.  Use of plant colonizing bacteria as chassis for transfer of N₂-fixation to cereals. , 2015, Current opinion in biotechnology.

[4]  V. Martin,et al.  Mining Enzyme Diversity of Transcriptome Libraries through DNA Synthesis for Benzylisoquinoline Alkaloid Pathway Optimization in Yeast. , 2016, ACS synthetic biology.

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

[6]  Tom Ellis,et al.  DNA assembly for synthetic biology: from parts to pathways and beyond. , 2011, Integrative biology : quantitative biosciences from nano to macro.

[7]  Christopher A. Voigt,et al.  Genomic Mining of Prokaryotic Repressors for Orthogonal Logic Gates , 2013, Nature chemical biology.

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

[9]  J. Lundeberg,et al.  Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing , 2013, Scientific Reports.

[10]  Robert P. St.Onge,et al.  Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples , 2010, Nucleic acids research.

[11]  Uriel Feige,et al.  Heuristic for maximizing DNA reuse in synthetic DNA library assembly. , 2014, ACS synthetic biology.

[12]  Michael Bissell,et al.  Genotype Specification Language. , 2016, ACS synthetic biology.

[13]  S. Weissman,et al.  Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Maxime Durot,et al.  Rapid and reliable DNA assembly via ligase cycling reaction. , 2014, ACS synthetic biology.

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

[16]  Yizhi Cai,et al.  Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units. , 2015, ACS synthetic biology.

[17]  Brian F. Pfleger,et al.  Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes , 2006, Nature Biotechnology.

[18]  J. McPherson,et al.  Coming of age: ten years of next-generation sequencing technologies , 2016, Nature Reviews Genetics.

[19]  F. Arnold,et al.  Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras , 2010, Current protocols in protein science.

[20]  Ryan T Gill,et al.  Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides , 2010, Nature Biotechnology.

[21]  L. Curatti,et al.  The Role of Nif Proteins in Nitrogenase Maturation , 2008 .

[22]  Duhee Bang,et al.  ‘Shotgun DNA synthesis’ for the high-throughput construction of large DNA molecules , 2012, Nucleic acids research.

[23]  R. Burris,et al.  In situ studies on nitrogen fixation with the acetylene reduction technique. , 1967, Science.

[24]  R. K. Meyer,et al.  The Coordinate-Exchange Algorithm for Constructing Exact Optimal Experimental Designs , 1995 .

[25]  Sven Panke,et al.  Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort , 2016, Nature Communications.

[26]  William C. Deloache,et al.  A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. , 2015, ACS synthetic biology.

[27]  D. G. Gibson,et al.  Enzymatic Assembly of Overlapping DNA Fragments , 2011, Methods in Enzymology.

[28]  Rahul Sarpeshkar,et al.  Synthetic analog computation in living cells , 2013, Nature.

[29]  Harold J. Kushner,et al.  A New Method of Locating the Maximum Point of an Arbitrary Multipeak Curve in the Presence of Noise , 1964 .

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

[31]  M. Elowitz,et al.  Combinatorial Synthesis of Genetic Networks , 2002, Science.

[32]  Lalit M. Patnaik,et al.  Adaptive probabilities of crossover and mutation in genetic algorithms , 1994, IEEE Trans. Syst. Man Cybern..

[33]  Huimin Zhao,et al.  Recent advances in DNA assembly technologies. , 2014, FEMS yeast research.

[34]  H. Salis,et al.  Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites , 2013, Nucleic acids research.

[35]  J. Kitzman,et al.  Massively Parallel Single Amino Acid Mutagenesis , 2014, Nature Methods.

[36]  Morten H. H. Nørholm,et al.  Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli. , 2015, ACS synthetic biology.

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

[38]  Steven Leonard,et al.  SASI-Seq: sample assurance Spike-Ins, and highly differentiating 384 barcoding for Illumina sequencing , 2014, BMC Genomics.

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

[40]  Allan Kuchinsky,et al.  The Synthetic Biology Open Language (SBOL) provides a community standard for communicating designs in synthetic biology , 2014, Nature Biotechnology.

[41]  Donald R. Jones,et al.  A Taxonomy of Global Optimization Methods Based on Response Surfaces , 2001, J. Glob. Optim..

[42]  Christopher A. Voigt,et al.  Genetic circuit design automation , 2016, Science.

[43]  C. Satishchandran,et al.  Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments. , 2000, Gene.

[44]  C. Tomlin,et al.  Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay , 2013, Nucleic acids research.

[45]  Matthew W Lux,et al.  Genetic design automation: engineering fantasy or scientific renewal? , 2012, Trends in biotechnology.

[46]  F. van Nieuwerburgh,et al.  Library construction for next-generation sequencing: overviews and challenges. , 2014, BioTechniques.

[47]  T. Mikkelsen,et al.  Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes , 2014, Nucleic acids research.

[48]  Huimin Zhao,et al.  DNA assembly techniques for next-generation combinatorial biosynthesis of natural products , 2014, Journal of Industrial Microbiology & Biotechnology.

[49]  Nicholas Roehner,et al.  Double Dutch: A Tool for Designing Combinatorial Libraries of Biological Systems. , 2016, ACS synthetic biology.

[50]  Zhen Zhang,et al.  Sharing Structure and Function in Biological Design with SBOL 2.0. , 2016, ACS synthetic biology.

[51]  Jed Dean,et al.  High-throughput, cost-effective verification of structural DNA assembly , 2013, Nucleic acids research.

[52]  G. Church,et al.  Accurate multiplex gene synthesis from programmable DNA microchips , 2004, Nature.

[53]  J. Rohr,et al.  Combinatorial biosynthesis of antitumor indolocarbazole compounds. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[54]  David Baker,et al.  Multiplex pairwise assembly of array-derived DNA oligonucleotides , 2015, Nucleic acids research.

[55]  Christopher A. Voigt,et al.  Characterization of 582 natural and synthetic terminators and quantification of their design constraints , 2013, Nature Methods.

[56]  Carola Engler,et al.  Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes , 2009, PloS one.

[57]  Timothy S. Ham,et al.  Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools , 2012, Nucleic acids research.

[58]  Jacob Beal,et al.  An end-to-end workflow for engineering of biological networks from high-level specifications. , 2012, ACS synthetic biology.

[59]  Christopher A. Voigt,et al.  Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca , 2012, Proceedings of the National Academy of Sciences.

[60]  H. Salis,et al.  Efficient search, mapping, and optimization of multi‐protein genetic systems in diverse bacteria , 2014 .

[61]  G. Stephanopoulos,et al.  Improving Metabolic Pathway Efficiency by Statistical Model-Based Multivariate Regulatory Metabolic Engineering. , 2017, ACS synthetic biology.

[62]  Christopher A. Voigt,et al.  Algorithmic co-optimization of genetic constructs and growth conditions: application to 6-ACA, a potential nylon-6 precursor , 2015, Nucleic acids research.

[63]  Maido Remm,et al.  Enhancements and modifications of primer design program Primer3 , 2007, Bioinform..

[64]  B. Faircloth,et al.  Primer3—new capabilities and interfaces , 2012, Nucleic acids research.

[65]  Jay Shendure,et al.  Parallel, tag-directed assembly of locally derived short sequence reads , 2010, Nature Methods.

[66]  A. Iafrate,et al.  Anchored multiplex PCR for targeted next-generation sequencing , 2014, Nature Medicine.

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

[68]  Zhen Zhang,et al.  libSBOLj 2.0: A Java Library to Support SBOL 2.0 , 2015, IEEE Life Sciences Letters.

[69]  Keith E. J. Tyo,et al.  Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli , 2010, Science.

[70]  Swapnil Bhatia,et al.  Functional optimization of gene clusters by combinatorial design and assembly , 2014, Nature Biotechnology.

[71]  Alistair Elfick,et al.  PaperClip: rapid multi-part DNA assembly from existing libraries , 2014, Nucleic acids research.

[72]  Jay Shendure,et al.  Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules , 2012, Nature Methods.

[73]  Jacob Beal,et al.  CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. , 2016, ACS synthetic biology.

[74]  Hal S Alper,et al.  Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. , 2013, Metabolic engineering.

[75]  Vivek K. Mutalik,et al.  Composability of regulatory sequences controlling transcription and translation in Escherichia coli , 2013, Proceedings of the National Academy of Sciences.

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

[77]  H. Bayley,et al.  Continuous base identification for single-molecule nanopore DNA sequencing. , 2009, Nature nanotechnology.

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

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

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

[81]  P. Ludden,et al.  Maturation of Nitrogenase: a Biochemical Puzzle , 2005, Journal of bacteriology.

[82]  Drew Endy,et al.  Engineering BioBrick vectors from BioBrick parts , 2008, Journal of Biological Engineering.

[83]  D. Densmore,et al.  Algorithms for automated DNA assembly , 2010, Nucleic acids research.

[84]  S. Beck.  Multiplex DNA sequencing. , 1993, Methods in molecular biology.