Michael Pallone
BIOL 509
December 8, 2010
Warner, J. R., Reeder, P. J., Karimpour-Fard, A., Woodruff, L. B. A., Gill, R. T. (August 2010).
Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides.
Nature Biotechnology 28(8):856-862.
Introduction:
Microbial genomes hold the potential for tremendous combinatorial diversity; however,
researchers’ ability to search through this diversity remains limited to the number of individuals which
can be tested. Thus, there is a high demand for strategies able to define relevant genetic variation then
thoroughly search through the diverse space. This issue has been studied at great depth at the level of
individual genes and advances in genomics, such as multiplex DNA synthesis and recombineering, now
allow the extension of these strategies to the genome scale.
These advances in genomics have also resulted in several methods which allow highly parallel
mapping of genes to traits. Molecular barcoding, one such tool, involves the replacement of every gene
in Saccharomyces cerevisiae with a specific DNA sequence which can then be tracked through
microarray. However, limitations exist such as the difficulty of creating new mutations. These limits
challenge efforts to apply these methods of phenotypic dissecting and reengineering reliant on the
coordinated actions of multiple genes and mutations.
Research over the past decade has resulted in recombination-based methods, recombineering,
that makes it easier to specifically modify the E. coli genome using synthetic DNA (synDNA). The current
study employs TRackable Multiplex Recombineering (TRMR), a versatile method that allows the
simultaneous creation and mapping of thousands of specific genetic modifications by combining parallel
DNA synthesis, recombineering and molecular barcoding technology.
Methods and Results:
A comprehensive library of synDNA cassettes containing a blasticidin-S resistance gene were
designed as one of two types. “Up” cassettes contain a strong and repressible PLtet-1 promoter along
with Ribosome Binding Site (RBS) sequences, both generally increasing downstream gene transcription
and translation. “Down” cassettes were designed to replace the RBS sequences with inert sequences,
this generally decreasing translation initiation. Molecular barcodes (“tags”) were incorporated to track
each synDNA oligonucleotide and to track each allele within the mixed population on a barcode
microarray. Targeting oligos were designed to insert upstream, replace the start codon, and account for
gene overlap. Targeting oligos were designed for every possible protein-encoding gene in the E. coli
genome. Promoter replacement for lacZ and galK genes were performed to test cassette construction
and to optimize the procedure for allele production. Alleles were isolated as colonies and all showed
the expected change in regulation and expression.
4,077 “Up” and “Down” oligos were constructed from the 8,154 targeting oligos in separate
pools. To confirm the desired mutation occurred, PCR amplification of 390 colonies was performed
followed by sequencing of the cassettes and neighboring chromosome DNA. 34 of 34 distinct alleles
showed the correct insertion of cassettes into the correct genome location with only 3 showing DNA
errors. This indicating that complex oligonucleotide mixtures may be used to engineer an identify
thousands of distinct genomic loci with decent accuracy. Further assessing of complete and uniform
library creation using multiplex was performed with Affymetrix Geneflex TAG4 arrays to measure
barcode concentration both before and after recombineering. Nearly complete (98%), up and down
oligo libraries were created; and, microarray analysis of cell mixtures showed a 96% creation of unique
alleles.
TRMR potential to rapidly generate and identify mutated alleles of interest was demonstrated
by plating mixtures on agar medium supplemented to create four different conditions (salicin, D-fucose,
methylglyoxal and valine) in which wild-type E. coli do not typically grow. Colonies representing resistant
mutants arose at frequencies greater than 100-fold higher than unmodified control cells, reliant on
spontaneous mutation, to generate resistance. TRMR performance at the genome scale was further
demonstrated by combining the “up” and “down” allele libraries and measuring their fitness in liquid
cultures containing rich or minimal nutrients. Simultaneous tracking of all 2,500 alleles, reduced from
growth selections, was successfully done using barcode microarrays.
Lastly, TRMR was used to identify genes that improve tolerance to lignocellulosic hydrolysate
derived from corn stover. Growth was measure for variants, containing the alleles from the prior
experiments, in several mixtures of hydrolysate and minimal medium. Microarray analysis of the alleles
indicated that only a small portion of the population remained after each selection. Modifications that
improved growth in the lower concentrations of corn stover hydrolysate were shown to affect genes
known to be involved in primary metabolism, RNA metabolism and sugar transport. Modifications that
improved growth in higher concentrations of hydrolysate selected for alleles related to secondary
metabolism, vitamin metabolism and antioxidant activity. These mutations conferring fitness
advantages were tested by the isolation selection and characterized growth of seven alleles in
hydrolysate relative to unmutated E. coli. All seven alleles yielded either higher growth rate or biomass
production relative to wild-type strains of E. coli.
Conclusions:
TRMR, which is versatile and easy to use, as demonstrated, can increase the throughput of
genetic studies by several orders of magnitude. TRMR is similar to genomic methods used by the yeast
community and is therefore conformational to a wide range of freely and commercially available
software packages; however, the acquisition of oligonucleotide libraries presents a primary challenge.
As DNA synthesis technologies continue to improve this challenge may become less of a problem.
Additionally, TRMR can be carried out recursively, allowing for the accumulation of multiple beneficial
mutations within a genome. Finally, integration of TRMR into directed-evolution programs could
provide genome-scale construction and tracking of mutational combinations, the ability to do which
would improve both the understanding and engineering of complex traits.