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 (beta-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.
Warner
Joseph R
JR
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado, USA.
Reeder
Philippa J
PJ
Karimpour-Fard
Anis
A
Woodruff
Lauren B A
LB
Gill
Ryan T
RT
D001483:Base Sequence; D019295:Computational Biology / Q000379:methods; D058893:DNA Barcoding, Taxonomic; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods; D016680:Genome, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
D029968:Escherichia coli Proteins; D009841:Oligonucleotides