16 2 Trackable Multiplex Recombineering (TRMR) and Next-Generation Genome Design Technologies
the desired phenotype. The field of genetics and the discovery of the mecha-
nisms by which traits are propagated along generations allowed people, for the
first time, to rationally induce genetic modifications rather than wait for them to
occur randomly. Early efforts focused on rational transfer and modifications of
single genes and were collectively termed “genetic engineering.” Complex traits,
however, derived from multiple gene interactions or whole metabolic pathways,
cannot be efficiently engineered one gene at a time and require high-throughput
and systemic approaches, the recognition of which formed the basis of the field
of metabolic engineering [1, 2].
Since then, advances in DNA sequencing and systems biology methodologies
have led to exceptional new approaches for characterizing complex traits and
their underlying genetic networks. Additionally, rapid progress in DNA chemical
synthesis and the development of recombination-based methods now allow
mutations to be incorporated in multiplex and at a throughput orders of magni-
tudes beyond the state of the art a decade ago [3–6]. In contrast to earlier meth-
ods of individually synthesizing oligonucleotides or using DNA segments from
natural sources, current technology allows the parallel production of synthetic
DNA (synDNA) libraries [5]. Additionally, homologous recombination-based
techniques (recombineering) that promote the integration of foreign DNA into
the chromosome of the target organism have reached relatively high levels of
efficiency [6]. Recombineering in E. coli is based on targeting a synthetic recom-
bineering substrate (a single-stranded (ss) DNA oligonucleotide or a double-
stranded (ds) DNA cassette) to a specific locus on the chromosome via homology
arms. Typically, this DNA substrate contains a desired mutation and may also
code for an antibiotic resistance gene as a selective marker. The actual recombi-
nation is enabled by either the Rec E/T or the λ-Red prophage system [6, 7].
Here, we describe the TRMR and T^2 RMR techniques, which not only make the
multiplexing of recombineering possible in E. coli but also provide the ability to
track the engineered genetic changes accurately. Currently, both library designs
allow one to target, in parallel, every gene in the genome for either overexpres-
sion or downregulation, with T^2 RMR allowing for tuning of gene expression over
a ~10^4 -fold range. The trackability is achieved by adding a unique “molecular
barcode” [8] upstream of every mutation, facilitating its identification. These
methods enable the search for specific and desired genetic traits and aid in the
navigation of an otherwise large mutational space (i.e., in this case, the total
number of possible single mutations). We discuss the benefits of such methods,
existing challenges, possible combinations with other methods, and some
possible future development and applications.2.2 Current Recombineering Techniques
E. coli did not evolve an efficient mechanism for recombination; therefore spon-
taneous homologous recombination of foreign genetic material is typically a rare
event, on the order of 10−6 for linear ssDNA or dsDNA substrates [9]. It has been
suggested that the low efficiency is primarily due to endogenous nucleases that
rapidly degrade the unprotected DNA [10, 11]. Phage-based methods, which rely