2.3 Trackable Multiplex Recombineering 23limitation is that the original TRMR libraries are binary in nature and do not
allow the tuning of expression levels. When engineering a synthetic pathway, the
activity of the pathway can be dependent on expressing each protein in a very
narrow expression range [28] rather than in a binary way.
The design of the shared DNA for T^2 RMR addresses these limitations.
T^2 RMR uses a “bicistronic design” (BCD), devised by Mutalik et al. [29], that
embeds two RBSs in a nucleotide sequence that encodes a 16-amino-acid pep-
tide, which is then placed directly in front of a gene of interest. The dual RBSs
embedded in this leader peptide affect ribosome binding and translation initia-
tion, and therefore these BCD constructs give much more consistent expres-
sion when tested with a variety of genes. Mutalik et al. further tested several
hundred promoter variants, as well as combinations of BCDs and promoters, to
express genes over a wide dynamic range. In 93% of cases, they found they could
predict the expression level of a protein to within twofold, which is a great
improvement over using a single RBS [29]. T^2 RMR consists of four libraries/
shared DNAs, each expressing a different BCD, to give four base expression
levels: “off,” “low,” “intermediate,” and “high.” Each library contains a 12-nucleo-
tide barcode to identify the BCD during high-throughput sequencing. An
inducible LacI-regulated promoter was placed in front of each BCD allowing
for fine-tuning of gene expression to almost any level that is desired just by
changing the amount of inducer (isopropyl β-d-1-thiogalactopyranoside
(IPTG)) that is added to the medium. Validation of T^2 RMR with the
β-galactosidase protein (lacZ gene) gave expression over a ~10^4 -fold activity
range, as measured by the Miller assay, by using different combinations of the
libraries and amounts of IPTG (Figure 2.4b).
2.3.2 Experimental Procedure
The experimental procedure is the same for both TRMR and T^2 RMR. Once the
double-stranded, linearized synDNA libraries have been constructed, they are
incorporated into the genome by homologous recombination. E. coli cells con-
taining the λ-Red recombination proteins (either integrated directly on the
chromosome or expressed from a plasmid such as pSIM5 [14]) are grown at
30 °C to mid-log phase in medium containing the appropriate antibiotic if
required. If using pSIM5, expression of the λ-Red enzymes is induced by incu-
bating cells at 42 °C for 15 min. The cells are then chilled on ice and made elec-
trocompetent by washing with ice cold water as previously described [11].
SynDNA is then transformed into cells by electroporation. After 2 h of recovery
at 37 °C, cells are spread on plates containing the antibiotic resistance marker
that is selective for library clones. Plates are incubated at 37 °C for 22 h and then
colonies are scraped from the plates, resuspended in LB medium, and aliquoted
for storage at −70 °C.
Screening and selection of TRMR and T^2 RMR clones can be carried out using
either liquid or solid media with any chemical compound that modifies growth
or confers a phenotype that can be detected by a high-throughput assay. An
equal number of cells from all libraries (either up and down or off, low, interme-
diate, and high) are mixed together in medium containing the antibiotic that is