title: Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies.
abstract: Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory time scales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites. This approach works by assembling genome engineered sites with cell-specific barcodes into a format compatible with high-throughput sequencing technologies. This approach, called barcoded-TRACE (bTRACE) was applied to assess E. coli populations engineered by recursive multiplex recombineering across both 6-target sites and 31-target sites. The 31-target library was then tracked throughout growth selections in the presence and absence of isopentenol (a potential next-generation biofuel). We also use the resolution of bTRACE to compare the influence of technical and biological noise on genome engineering efforts.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Colorado , 596 UCB Boulder, Colorado 80303, United States.
authors.forename: Ramsey I
authors.identifier: 0000-0003-2306-9206
authors.initials: RI
authors.lastname: Zeitoun
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Colorado , 596 UCB Boulder, Colorado 80303, United States.
authors.forename: Gur
authors.identifier: 0000-0002-1757-6722
authors.initials: G
authors.lastname: Pines
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Colorado , 596 UCB Boulder, Colorado 80303, United States.
authors.forename: Willliam C
authors.initials: WC
authors.lastname: Grau
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Colorado , 596 UCB Boulder, Colorado 80303, United States.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
mesh_terms: D000465:Algorithms; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering; D016680:Genome, Bacterial; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
keywords: DNA assembly; genome engineering; high throughput sequencing; synthetic biology