Synthetic Biology Parts, Devices and Applications

20 2 Trackable Multiplex Recombineering (TRMR) and Next-Generation Genome Design Technologies

into cells (Figure 2.3). During amplification, circular concatemers are created.

These concatemers are then cleaved by restriction enzyme digest to generate

linear dsDNA with the homology regions, molecular barcodes, antibiotic resist-

ance, and gene modification sequences in the correct order. The synDNA cas-

settes are linearized because linear DNA, and not circular DNA, is generally

used as a substrate for recombineering using the λ-Red system [6].

TRMR T2RMR

(a)

Engineered genomes

Improved genomes

Illumina MiSeq for

molecular barcodes

Genome plot of fitness

HA1P HA2

LacIO

GBTs BCD

Terminator

KanR Stop Tp Pi

Off LowIntermediateHighgeneA

geneB

geneA geneB

Off Low LowIntermediate

(b)

Engineered genomes

Microarray for

each gene

Improved genomes

Genome plot of fitness

BlastR

geneAgeneBgeneCgeneD

geneAgeneBgeneCgeneD

geneAgeneBgeneCgeneD

“Up” microarray

“Up” microarray

“Down” microarray

geneAgeneBgeneCgeneD

“Down” microarray

HA1PG Up/downHA2

Figure 2.2 Overview of trackable multiplex recombineering (a) and tunable trackable

multiplex recombineering (b). TRMR and T^2 RMR cassettes are designed and synthesized in

multiplex followed by transformation into Escherichia coli. The E. coli population is then placed

under selective pressure. Alleles that are enriched during selection are identified by

microarray (TRMR) or next-generation sequencing (T^2 RMR), and their relative fitness is

determined. Cassette design for each technique is shown at the top. Black regions are shared

DNA and gray regions are from the targeting oligos. HA1 and HA2, homology regions; P,

barcode priming site; G, barcode identifying the gene; B, barcode identifying the BCD; BlastR,

blasticidin resistance gene; KanR, kanamycin resistance gene; stop, three frame stop codons;

Ts, terminator spacer; Tp, terminator pause; Pi, promoter insulator; LacIO, LacI-regulated

synthetic inducible promoter (apFAB906); BCD, bicistronic design (dual RBS). Adapted with

permission from Freed et al. 2015 [22]. Copyright 2015 American Chemical Society.