Biology Reference
In-Depth Information
Saccharomyces cerevisiae
, an entire biochemical pathway can be constructed in a single step.
This tool has been directly applied to pathways up to 45.3 kb in length with high efficiency.
As proof of concept, the biosynthetic pathway of a rare nitroaryl-substituted polyketide,
aureothin, was assembled into
S. cerevisiae
. Seven fragments with sizes ranging from 4 to
5 kb were amplified and cotransformed with the three helper fragments to make a single
circular DNA molecule of 35.7 kb. Six out of ten randomly picked colonies were confirmed
as correct by restriction digestion, which indicates 60% efficiency.
The isothermal assembly method developed by Gibson et al. uses an in vitro recombination
system to assemble overlapping DNA fragments.
24,25
An enzyme cocktail containing
T5 exonuclease, Phusion DNA polymerase, and
Taq
DNA ligase executes overhang chew-
back, gap-filling, and ligation all in one step. Gibson et al. reported the construction of a
16.3 kb mouse mitochondrial genome from 600 overlapping 60-mers in vitro,
demonstrating that this method is a successful approach for multiple-fragment, multiple-
size assembly.
25
Employing the same method, Smith et al. reported a one-step
combinatorial assembly of promoter and gene cassettes in
E. coli
. An acetate utilization
pathway containing one acetate kinase gene and one phosphotransacetylase gene was
constructed, and a synthetic library containing three promoters and four gene variants was
constructed with 81% assembly efficiency.
24
Ordered Gene Assembly in
Bacillus subtilis
(OGAB) is a one-step assembly method to
combine multiple DNA fragments into a
B. subtilis
plasmid.
26,27
DNA fragments with
overhanging sequences generated by Type IIS restriction enzymes are amplified and ligated
by T4 DNA ligase in a specified order. In 2003, Itaya et al. successfully assembled five
antibiotic resistance genes in a designed order and orientation.
27
Later, the zeaxanthin
operon consisting of five carotenoid biosynthetic genes
crtE
,
crtY
,
crtI
,
crtB
, and
crtZ
was
assembled into a plasmid by the OGAB method, proving the capability of OGAB to
construct pathways in one step.
26
48
MULTISTEP CONSTRUCTION TOOLS
The BioBrick
TM
method is used to construct a metabolic pathway in a step-by-step manner.
Briefly, restriction digestions and ligations are implemented with compatible sticky ends
generated by a set of specific restriction enzymes
Eco
RI,
Xba
I,
Spe
I, and
Pst
I. The standard
prefix and suffix sequences containing corresponding restriction sites are cleaved by
Eco
RI and
Spe
I, or
Eco
RI and
Xba
I. During the construction step, since the
Xba
I recognition site TCTAGA
shares the overhang sequence CTAG with the
Spe
I recognition site ACTAGT, the two digested
sticky ends fuse together to generate a 6-bp scar sequence ACTAGA between the original
sequence and the inserted sequence.
28
Based on this method, the BglBrick method was
developed in which
Xba
I,
Spe
I, and
Pst
I are replaced with
Bgl
II,
Bam
HI, and
Xho
I, respectively,
thus alternating the scar sequence, along with the removal of a possible in-frame stop
codon.
29
These two approaches allow operon construction, serving as building blocks for
pathway assembly. However, the time-consuming manipulation and the existence of a scar
in key locations prevent its wide application in pathway construction.
Based on Type IIS restriction enzymes, a series of pathway construction tools have been
developed such as pairwise selection assembly method,
30
Golden Gate assembly method,
31
and Golden Braid method.
32
Among the methods utilizing Type IIS restriction enzymes,
Golden Gate assembly method attracts much attention because it can rapidly assemble
multiple DNA fragments in a one-pot, one-step manner. Type IIS enzymes recognize
asymmetric sequences and cleave outside of these recognition sites, resulting in overhangs
that can be ligated by a DNA ligase. Using the Golden Gate method, a model protein
trypsinogen was efficiently integrated into a vector.
33
Overlap extension polymerase chain reaction (OE-PCR) is a scarless method for DNA
assembly.
34
Primers are designed to contain sequences identical to the upstream and
