Biomedical Engineering Reference
In-Depth Information
designated LIC-PCR [
3
], LIC [
4
], and enzyme-free cloning [
5
]
use complementary single-strand overhangs to combine the insert
and the linearized vector.
In recent years along with the growing interest in synthetic
biology several tools were developed for integration of multiple
DNA fragments. The Sequence and Ligation-Independent Cloning
(SLIC, Chapter
2
) is based on homologous in vitro recombination
and single-strand assembly [
6
,
7
]. The SLIC was used for tandem
assembly of up to ten DNA fragments, simultaneously. Additional
approach is the Gibson Assembly™ (Chapter
1
)
(New England
Biolabs), which joins together multiple 3
overhangs DNA frag-
ments. The DNA assembly method was used to join and clone
DNA fragments up to several hundred kilobases in
E. coli
[
8
].
In this chapter on the Restriction-Free (RF) methodology and
in the following chapter on the Transfer-PCR (TPCR) platform we
describe DNA assembly methods based on whole plasmid amplifi -
cation of the vector and DNA insert(s). In both approaches gene
amplifi cation is carried out by two primers each containing at the
3
′
an extension, over-
lapping with the sites of integration in the destination vector [
9
,
10
]. Whereas, the RF methodology requires purifi cation of the
intermediate PCR product generated in the fi rst amplifi cation
stage for integration into the destination vector [
9
,
11
], in the
TPCR approach the synthesis of the PCR product and its assembly
into the destination vector occurs in a single-tube [
10
]. A useful
web tool was developed to facilitate the design of the primers for
cloning using the RF and TPCR methods [
12
]. Both methods
were used for various DNA manipulation applications, including
cloning and single- and multiple-site-targeted mutagenesis.
We have demonstrated the strength of the RF cloning not only
for single DNA fragment integration but also for simultaneous
multiple DNA fragments [
9
]. In the current protocol we describe
a detailed procedure for implementation of the RF cloning for
simultaneous multicomponents DNA assembly at distinct posi-
tions or in tandem within a circular expression vector of choice.
′
a target-gene-specifi c sequence and at the 5
′
2
Materials
2.1
Bacterial Strains
For DNA cloning and plasmid preparation procedures,
E. coli
DH5
(Agilent Technology, Stratagene division, Santa Clara, CA)
was used. For protein expression
E. coli
BL21(DE3) (Novagen/
EMD Millipore Chemicals, Darmstadt, Germany) was employed.
α
2.2
Vectors
For cloning experiments described in the methods section the expression
vectors pET21a and pACYCDuet-1 (Novagen/EMD Millipore
Chemicals, Darmstadt, Germany) were used as destination vectors.
