Synthetic Biology Parts, Devices and Applications

rahS

AND de

iii iii iv

Target

oligos

2 mixtures of

target oligos

2 mixtures of 4077

synDNA oligos

geneX up

geneY up

geneZ up

geneX

Shared DNA geneX^ up
X UP X
Y UP Y
Z UP Z
X DOWN X
Y DOWN Y
(a) ZDOWN Z

hS

dera

• AND

ffo

i ii iii iv

Target

oligos

4 mixtures of 4497

1 mixture of synDNA oligos

target oligos

4 shared DNAs

geneX

geneY

geneZ

Shared DNA

geneX

X HIGH X

Y HIGH Y

Z HIGH Z

X INT X

Y INT Y

Z INT Z

X LOW X

Y LOW Y

Z LOW Z

X OFF X
Y OFF Y
Z OFF Z
(b)

~4000 genes

targeted

simultaneously

Up

up

up

Up

Up

Chromosome

Chromosome

Chromosome

Chromosome

Recombineering enzymes

geneX

geneX

geneY

geneY geneZ

geneZ

geneX geneY geneZ
geneX geneY up geneZ
(c)

down

geneYdown

geneZdown

Figure 2.3 Construction and incorporation of TRMR (a) and T^2 RMR (b) cassettes. In both TRMR and T^2 RMR, targeting oligos
are ligated with a shared DNA cassette encoding a specific genetic function. The ligated synDNA cassettes are amplified by
rolling circle amplification and then cleaved to create a linear dsDNA substrate. (c) The linear synDNA cassettes are
recombineered into cells, targeting all genes at one time. Adapted from Warner et al. 2010 [26].