46
A. Meyerdierks and F.O. Glöckner
Large DNA fragments can be cloned into a variety of vectors (Green et al. 1997,
Tao and Zhang 1998). Among these, three are commonly used for the construction of metagenomic libraries: cosmid, fosmid and bacterial artificial chromosome
(BAC) vectors. Cosmid vectors are conventional plasmids in which one or two bacteriophage λ cos sites have been integrated (Collins and Hohn 1978). This allows
highly efficient in vitro packaging of the DNA with bacteriophage λ packaging
extracts. The average size of DNA fragments that can be cloned in cosmid vectors
is between 30 and 45 kbp. This is dependent on the size of the vector. The combined size of vector plus insert needs to be 78–105% of an average bacteriophage λ
genome in order to be suitable for DNA packaging into bacteriophage λ phage heads
(Sambrook and Russel 2001). Once in the host cell, cosmids are present at a high
copy number. This facilitates further screening of the libraries (Collins and Hohn
1978, Sambrook and Russel 2001). Cosmids have been used in the construction of
several metagenomic libraries (Entcheva et al. 2001, Piel 2002, Courtois et al. 2003,
Schmeisser et al. 2003, Sebat et al. 2003, Lopez-Garcia et al. 2004). However, the
system has two major drawbacks. Firstly, chimeras, rearrangements, and deletions
have been observed (Monaco and Larin 1994), secondly the insert sizes are more or
less uniform, but relatively small compared to a complete genome.
Fosmid vectors (Kim et al. 1992) are currently the most popular vectors for
metagenome analyses (e.g., Stein et al. 1996, Schleper et al. 1997, 1998, Beja et al.
2002, Quaiser et al. 2002, 2003, Meyerdierks et al. 2005, DeLong et al. 2006,
Neufeld et al. 2008). Fosmid cloning also takes advantage of the in vitro packaging
system for bacteriophage λ, resulting in insert sizes similar to those obtained with
cosmid vectors. In contrast to cosmid vectors, however, fosmid vectors are derived
from the E. coli F(ertility)-factor and carry the replication and partition sequences
of the F-factor plasmid. As a result, there are only 1–2 fosmid copies per cell and
two different fosmids cannot be maintained in a single cell. The expression of toxic
gene products that could be lethal for the host is reduced due to the lower copy
number, and chimeras are less frequent. To facilitate screening, improved fosmid
vectors have been constructed, which carry a second inducible replicon (Wild et al.
2002). This allows maintenance of the library in the low copy state, and the selective induction of 10–50 copies per cell for screening and further analysis. With these
improved vectors fosmid cloning and screening is straightforward. This may be the
reason for the success of these vectors in metagenome analyses.
BAC vectors (Shizuya et al. 1992) have been developed to overcome the insert
size limitation of fosmids, intrinsic to the bacteriophage λ packaging system. BAC
vectors, like fosmid vectors, carry the F-factor replication and partition sequences
from E. coli. and are also available with a second inducible replicon (Handelsman
et al. 2002, Wild et al. 2002). The main difference between these two cloning systems is the method used to introduce the recombinant DNA into the host. In contrast
to fosmid cloning, electroporation is used for BAC library construction, to circumvent the size limitation of the bacteriophage λ packaging system. Specific E. coli
host strains are used which have been shown to transform well with large insert
constructs (Sheng et al. 1995). It has been shown that DNA fragments larger than
300 kbp can be cloned into BAC vectors and stably maintained in E. coli (Shizuya
A. Meyerdierks and F.O. Glöckner
Large DNA fragments can be cloned into a variety of vectors (Green et al. 1997,
Tao and Zhang 1998). Among these, three are commonly used for the construction of metagenomic libraries: cosmid, fosmid and bacterial artificial chromosome
(BAC) vectors. Cosmid vectors are conventional plasmids in which one or two bacteriophage λ cos sites have been integrated (Collins and Hohn 1978). This allows
highly efficient in vitro packaging of the DNA with bacteriophage λ packaging
extracts. The average size of DNA fragments that can be cloned in cosmid vectors
is between 30 and 45 kbp. This is dependent on the size of the vector. The combined size of vector plus insert needs to be 78–105% of an average bacteriophage λ
genome in order to be suitable for DNA packaging into bacteriophage λ phage heads
(Sambrook and Russel 2001). Once in the host cell, cosmids are present at a high
copy number. This facilitates further screening of the libraries (Collins and Hohn
1978, Sambrook and Russel 2001). Cosmids have been used in the construction of
several metagenomic libraries (Entcheva et al. 2001, Piel 2002, Courtois et al. 2003,
Schmeisser et al. 2003, Sebat et al. 2003, Lopez-Garcia et al. 2004). However, the
system has two major drawbacks. Firstly, chimeras, rearrangements, and deletions
have been observed (Monaco and Larin 1994), secondly the insert sizes are more or
less uniform, but relatively small compared to a complete genome.
Fosmid vectors (Kim et al. 1992) are currently the most popular vectors for
metagenome analyses (e.g., Stein et al. 1996, Schleper et al. 1997, 1998, Beja et al.
2002, Quaiser et al. 2002, 2003, Meyerdierks et al. 2005, DeLong et al. 2006,
Neufeld et al. 2008). Fosmid cloning also takes advantage of the in vitro packaging
system for bacteriophage λ, resulting in insert sizes similar to those obtained with
cosmid vectors. In contrast to cosmid vectors, however, fosmid vectors are derived
from the E. coli F(ertility)-factor and carry the replication and partition sequences
of the F-factor plasmid. As a result, there are only 1–2 fosmid copies per cell and
two different fosmids cannot be maintained in a single cell. The expression of toxic
gene products that could be lethal for the host is reduced due to the lower copy
number, and chimeras are less frequent. To facilitate screening, improved fosmid
vectors have been constructed, which carry a second inducible replicon (Wild et al.
2002). This allows maintenance of the library in the low copy state, and the selective induction of 10–50 copies per cell for screening and further analysis. With these
improved vectors fosmid cloning and screening is straightforward. This may be the
reason for the success of these vectors in metagenome analyses.
BAC vectors (Shizuya et al. 1992) have been developed to overcome the insert
size limitation of fosmids, intrinsic to the bacteriophage λ packaging system. BAC
vectors, like fosmid vectors, carry the F-factor replication and partition sequences
from E. coli. and are also available with a second inducible replicon (Handelsman
et al. 2002, Wild et al. 2002). The main difference between these two cloning systems is the method used to introduce the recombinant DNA into the host. In contrast
to fosmid cloning, electroporation is used for BAC library construction, to circumvent the size limitation of the bacteriophage λ packaging system. Specific E. coli
host strains are used which have been shown to transform well with large insert
constructs (Sheng et al. 1995). It has been shown that DNA fragments larger than
300 kbp can be cloned into BAC vectors and stably maintained in E. coli (Shizuya