248 12 Metabolic Channeling Using DNA as a Scaffold
arrangement of the DNA‐binding proteins along the DNA (Figure 12.1d). The
main twist comes with the requirement that each of these DNA‐binding pro
teins/domains is fused to a different functional protein. Therefore, the sequence
of target motifs encoded by the DNA program also defines the arrangement of
those functional proteins along with the order of the DNA‐binding domains.
Only by changing the sequence of a DNA program, either switching positions
or adding new target sequences, can outcome be predicted in advance (Figure
12.1d). This requires a method for the site‐specific targeting of enzymes along
the DNA surface. While there are 64 nucleotide triplets in the natural code for
the 20 amino acids, there could be as many as 262,144 different motifs consist
ing of nine nucleotides. Zinc fingers [19] and TAL elements [20] can be designed
to bind to almost any desired nucleotide sequence, ranging from 9 to as many
as 18 nucleotides. Additionally, we can select the target nucleotide sequence for
each available DNA‐binding protein.12.3.1 Zinc Finger Domains
There are more than 700 experimentally characterized zinc fingers in the data
base ZIFDB [21], offering a huge selection of building elements for synthetic
biology [22, 23]. Moreover, zinc fingers have similar properties, such as binding
affinity or stability, which is important, since we do not need to adjust the prop
erties of each separated part. The DNA program, therefore, represents a modu
lar approach for various synthetic biology applications.
Up until now, only zinc finger DNA‐binding domains were used to link bio
synthetic proteins to DNA scaffolds. Conrado et al. used five different zinc fin
ger domains (PBSII, Zif268, ZFa, ZFb, and ZFc) that were each comprised of
three fingers, with a specificity for unique 9‐bp DNA sequences [11, 24–26]
(Table 12.2). Statistically, a 9‐bp‐long sequence could appear 1.2 times per
genome in E. coli, if we assume that the nucleotide sequence distribution within
the genome is random. Lee et al. [10] used ADBs with four fingers that recog
nized a 12‐bp DNA sequence. All of the zinc fingers used were relatively short
and bound the DNA with low nanomolar affinity. Crucially, the selected zinc
finger domains should not bind functional regions of essential genes in E. coli or
affect bacterial fitness.
As an in vitro test of the system components, binding to DNA can be analyzed
using surface plasmon resonance (SPR) [27] (Figure 12.5a) or split GFP technol
ogy [28]. The DNA binding of the candidate zinc finger domains can be fused
with split fluorescent proteins. Reassembly of the split yellow fluorescent protein
(YFP) and strong fluorescence indicative of YFP reassembly occur only in the
presence of a DNA scaffold that contains neighboring binding sites for, for exam
ple, PBSII and Zif268, separated by only 2 bp (Figure 12.5b) [11].
To investigate whether zinc finger domains bind their cognate DNA targets
in vivo, a simple β‐galactosidase test for DNA‐binding domain activity in E. coli
was used (Figure 12.5c). The principle of this test is that an active zinc finger
domain should bind to its specific target sequence in the PSYN promoter and act
as a synthetic repressor, thereby decreasing the basal activity of this promoter
and lowering β‐galactosidase levels.