Synthetic Biology Parts, Devices and Applications

22 2 Trackable Multiplex Recombineering (TRMR) and Next-Generation Genome Design Technologies

In both TRMR and T^2 RMR, targeting oligos were designed using homology

regions that result in the synDNA cassette being inserted upstream of each gene

and replacing each gene’s start codon in E. coli MG1655. In the original demon-

stration of TRMR [26], all protein-coding genes were targeted. Each targeting

oligo also contained a unique 20-nucleotide sequence that served as a molecular

barcode (or “tag”) used to track each gene. The barcodes were chosen from a set

that had previously been used successfully in yeast [8]. In T^2 RMR [22], pseudo-

genes and noncoding RNAs were targeted in addition to all protein-coding

genes. Each targeting oligo contained a 12-nucleotide sequence that had been

optimized to serve as a molecular barcode for high-throughput sequencing.

Targeting oligos for both TRMR and T^2 RMR were synthesized on a microchip by

Agilent.

The most significant differences between TRMR and T^2 RMR are in the design

of the shared DNA. TRMR consists of two libraries: an “up” library that causes

genes to be overexpressed and a “down” library that causes genes to be underex-

pressed. The shared DNA for the up cassette contains the strong PLtetO-1 pro-

moter and a strong ribosome binding site (RBS), which generally results in

increased transcription and translation of downstream genes. The shared DNA

for the down cassette contains no promoter and no RBS, resulting in the deletion

of the native RBS and subsequent decrease in translation initiation. The activity

of the β-galactosidase protein (lacZ gene) was used to confirm that the up con-

struct for this protein resulted in overexpression and the down construct resulted

in loss of expression of the protein (Figure 2.4a).

While these libraries have been successful in identifying alleles responsible for

a desired phenotype, there are some limitations to the original TRMR libraries.

One drawback is that these libraries do not use standardized synthetic parts,

which may result in inconsistent expression levels across targeted genes, since it

is known that placing the same promoter or RBS in front of two different genes

can cause the two genes to be expressed at vastly different levels [22–25]. Another

Intermediate High

MG1655 Off Low

(b)

Modified Miller Units (log)

0 0.01 0.125 1

100000

10000

1000

100

10

1

IPTG (M)

Library

(a)

IacZ up

Wild type

IacZ down

Wild type

Glucose + X-gal IPTG + X-gal

Figure 2.4 Validation of TRMR (a) and T^2 RMR (b) cassettes using the lacZ gene. In TRMR, the

“up” cassette causes lacZ to be expressed, while the “down” cassette results in a loss of

expression. In T^2 RMR, varying the four libraries (“off,” “low,” “intermediate,” and “high”), and the

amount of inducer (IPTG) allows lacZ to be expressed over a ~10^4 -fold range. Adapted with

permission from Warner et al. 2010 [26] and Freed et al. 2015[22]. Copyright 2015 American

Chemical Society.