Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides

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Abstract

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.

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Figure 1: TRMR method.
Figure 2: Multiplex strategy to rapidly generate cell mixtures with defined genetic modifications.
Figure 3: Analysis of synDNA and cell library.
Figure 4: Trait-conferring genotypes identified in four selective environments.
Figure 5: Alleles identified during pooled growth in media and cellulosic hydrolysate.

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Acknowledgements

We thank D. Court (Center for Cancer Research, National Cancer Institute at Frederick, Maryland) for sharing plasmid pSIM5, C. Nislow and G. Giaever (University of Toronto, Ontario) for help with microarray analysis, A. Mohagheghi and M. Zhang (US National Renewable Energy Laboratories) for hydrolysate samples, M. O'Donnell for help in preparation of selective agar plates, Agilent for access to the Oligonucleotide Library Synthesis product, and H. Marshall and the University of Colorado Microarray Facility for molecular barcode genotyping. The authors appreciate financial support provided by Shell, the Colorado Center for Biorefining and Biofuels (http://www.C2B2web.org) and the Colorado Energy Initiative (http://rasei.colorado.edu).

Author information

J.R.W. and R.T.G. conceived the study; J.R.W. designed and performed all experiments except for growth selections and allele confirmations in hydrolysate, which were conducted by P.J.R.; A.K.-F. aided J.R.W. in selection of targeting sequences and selection of barcode tags; A.K.-F. and P.J.R. assigned gene ontology terms; L.B.A.W. aided J.R.W. in selection design and microarray analysis; L.B.A.W. constructed circle plots; P.J.R., A.K.-F. and L.B.A.W. helped in manuscript preparation; J.R.W. and R.T.G. wrote the manuscript; R.T.G. supervised all aspects of the study.

Correspondence to Ryan T Gill.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Tables 2–6, Supplementary Notes and Supplementary Figs. 1–4 (PDF 5193 kb)

Supplementary Table 1

Targeting oligo sequences, tag sequences, and 2-state hybridization energies. (XLS 2731 kb)

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