Page 35 - Macromolecular Crystallography

24 MACROMOLECULAR CRYS TALLOGRAPHY

et al., 2002). However, most projects have adopted integration host factor (IHF) and the att recombina-
LIC for the obvious reason that it is independent tion sequences attached to the DNA to be cloned.
of the input sequence. The three methods that are Directional cloning of the DNA insert is ensured
commercially available are described below. These by using two nearly identical but non-compatible
methods are generally carried out in 96-well format versions of the λ att recombination site (Fig. 2.1b).
andarethereforeamenabletolaboratoryautomation Expression vectors are usually constructed in two
using standard liquid handling systems. stages. In the first step an entry (a.k.a. master
or capture) clone is generated using recombina-
tion between attB and attP sites in the input DNA,
2.2.1 Ligation-independent cloning methods
usually a PCR product, and the donor vector respec-
2.2.1.1 LIC-PCR tively (BP reaction). The inserted DNA can then
Ligation-independent cloning of PCR products be transferred to one or more destination vectors
(LIC-PCR) was developed over 10 years ago to generate expression clones (LR reaction). The
(Aslandis, 1990; Haun et al. 1992). It is based on ability to generate rapidly and with high efficiency
the use of T4 DNA polymerase in the presence of multiple expression vectors with different formats
a single deoxyribonucleotide to produce 12–15 bp (e.g. fusion tags) from the same starting vector
overhangs in a PCR product that are complemen- is a unique property of the system. To select for
tary to sequences generated in the recipient vec- the desired recombinants and against parental plas-
tor (Fig. 2.1a). These extensions anneal sufficiently mids, in both BP and LR steps, the Gateway™ sys-
strongly to allow transformation of E. coli without tem uses the E. coli lethal gene ccdB in combination
the need to ligate the fragments, which is carried with differential antibiotic-resistance markers on the
out by repair enzymes in the host. Advantages of entry and destination plasmids. The Gateway™
the LIC-PCR system are that it does not require method is shown schematically in Fig. 2.1b and
specialized vectors and the reagents are relatively detailed protocols are available from the manufac-
inexpensive. However, the system does require turer (www.invitrogen.com).
the preparation of a high-quality, linearized vector, The use of this method of ligation-independent
which will require batch checking to ensure high cloning has been reported by several large-scale
efficiency of cloning. A limitation of the LIC-PCR is cloning projects (Luan et al., 2004; Abergel, 2003;
that one of four bases has to be preselected as the Vincentelli et al., 2003). In general, it appears that
‘lock’ in the compatible overhangs and hence the the BP reaction is largely insensitive to the con-
base pair composition of the annealing regions is centration of input PCR product and for ORFs
limited to using the other three bases. Consequently, <2 kb, cloning efficiency yields of nearly 90% can
the method is not entirely sequence independent be obtained (Marsischky and LaBaer, 2004). For
and cannot be used to join any sequence to any larger inserts (2–3 kb) a 50% drop in yield has been
other sequence. However, by appropriate vector reported (Marsischky and LaBaer, 2004). Ease of use
design LIC-PCR has been successfully implemented comes at a price since the 28–31 bp att sequences
in HTP mode (Stols et al., 2002). A protocol for car- add to the cost of the primers and the recombina-
rying out LIC-PCR using a commercially available tion enzymes – BP clonase (λ integrase + E. coli IHF)
vector system (www.novagen.com) is described in and the LR clonase (λ excisionase + λ integrase +
Protocol 2.1. E. coli IHF) – are relatively expensive compared to
standard DNA-modifying enzymes. Consequently,
2.2.1.2 Gateway™ we and others (Braun et al., 2002) have modified the
Gateway™ cloning technology is a modification of standard protocol by halving the recommended vol-
the recombination system of phage λ (Walhout et al., ume of reagents for both BP and LR steps, hence
2000; Hartley et al., 2000). The Gateway™ system reducing the final reaction volume to 10 µl without
utilizes a minimum set of components of the λ loss in performance. In using the Gateway™ system,
system for in vitro transfer of DNA, namely the λ it is important to be aware of the effect the att recom-
integrase protein, λ excisionase, the E. coli protein bination sequences may have on expression and/or