2.3 Trackable Multiplex Recombineering 19process results in two daughter cells, one of which harbors the desired genetic
modification, while the other remains genetically identical to its parental ances-
tor, limiting this method to a theoretical maximum efficiency of 50% [15].
2.3 Trackable Multiplex Recombineering
The E. coli genome consists of over 4000 genes. When engineering the E. coli
genome for a desired trait (e.g., tolerance to a growth condition or increased
production of a valuable chemical), combinations of multiple genetic modifica-
tions are often required to achieve optimal performance. The result is a combi-
natorial mutation space that expands exponentially with the number of targeted
genes and quickly exceeds the size of space that can be searched on laboratory
time scales. For example, if each of the 4000 genes is modified to both an “off ”
and an “on” state, there are 2^4000 possible states. TRMR and T^2 RMR provide a
rapid and efficient way to modify an entire genome in a controlled manner and
to evaluate the effects of those genetic modifications simultaneously. Using these
techniques it is possible to modify >95% of the genes in E. coli in a single day. An
overview of the TRMR and T^2 RMR techniques is shown in Figure 2.2. In order
to engineer a genome using TRMR or T^2 RMR, a synDNA cassette is created
that encodes for a genetic feature (such as the overexpression or underexpression
of each specific gene) and a molecular barcode that is used to track each feature.
These synDNA cassettes are then introduced in parallel into cell populations via
recombineering. Next, the modified populations are grown in any desired growth
condition or in selective medium. Microarray or sequencing analysis of the
molecular barcodes is used to determine the relative fitness of each allele/
engineered cell in the surviving population under the chosen conditions. TRMR
and T^2 RMR libraries must be used to evaluate a phenotype that can be either
selected or screened for.
To date, TRMR and T^2 RMR have been used to map genes required for growth
in various types of media and to optimize tolerance to acetate, low pH, cellulosic
hydrolysate, isobutanol, ethanol, isopentenol, furfural, and various antibiotics
[22–26]. These studies have given insight into carbon source and vitamin utiliza-
tion, primary and secondary metabolism, and mechanisms of toxicity under a
variety of conditions.
While the next few paragraphs will provide basic information on the TRMR
and T^2 RMR methods, readers are referred to an in-depth protocol for complete
experimental details of TRMR [27].
2.3.1 TRMR and T^2 RMR Library Design and Construction
All TRMR and T^2 RMR libraries consist of two main parts: (i) “targeting” oligos
that contain homology to each gene in the E. coli genome, a molecular barcode
to identify each oligo uniquely, and sequences used to amplify each region of the
oligo by PCR, and (ii) “shared DNA” that encodes for a genetic function and an
antibiotic resistance marker. These two parts are then ligated to each other to
create synDNA cassettes that can then be amplified, linearized, and transformed