15 Cloning Vectors
233
sertion of foreign DNA into the MCR will cause the infected cells to lose
their ß-galactosidase activity thus forming clear plaques. This allows for
a color distinction between cells infected by wild-type phage versus recombinant phage, forming clear or blue plaques, respectively. M13mp18
and M13mp19 differ with respect to the order of restriction sites within
the MCR.
Cosmids
Cosmids originate from the plasmid pBR322, but are inserted into cells by
transfection. To understand their function, we have to focus once again on
the replication of Iambda phage (Figure 4).
It is a typical feature ofA.-replication, that the phage DNAs areformedas one
long, so-called concatemeric molecule. Multiplegenomesare joined endto-end, separated by cohesive sites ( cos sites ). Cos sites are single-stranded,
complementary regions that are recognized by the packaging enzymes. The
DNA between cos sites is packaged into a phage particle as long as the distance between the two sites is in the range of 35-45 kb. This mechanism
provides the functional basis for cosmids (cos-site; plas-mid) (Collins
1979). pBR322 was changed so that a cos site was inserted into the tetracycline-resistance gene. Linearized plasmids will form concatameres similiar to those described for A.-replication. These cosmids arenot infectious,
since they Iack the phage's own coding sequences. However, in the presence
of enzymes ("packaging extract") and Iambda particles, in vitro packaging
of the recombinant DNA is efficient as long as the insert does not exceed[K.D.6] 35-45 kb in length. Cosmids replicate in the cell via the origin
of replication of pBR322 and may be isolated using the techniques described
for plasmids. Cosmid vectors are useful for genomic library construction
(Meyerowitz 1980).
Host cells
All ofthe host cells (Bachmann 1972, Bachmann 1980, Sambrook 1989) described below are derived from E. coli. Most of the vectors require specific
bacterial strains. For example, the use ofvectors expressing ß-galactosidase
gene activity requires the use oflac-, pro- bacterial strains, such as JM 103 ( see
M13 derivaties). A modern bacterial strain fulfilling this task is XL1-blue™
(Stratagene). Furthermore this strain can be grown on complete medium,
forms sex-pili and carries tetracycline-resistance-gene.
233
sertion of foreign DNA into the MCR will cause the infected cells to lose
their ß-galactosidase activity thus forming clear plaques. This allows for
a color distinction between cells infected by wild-type phage versus recombinant phage, forming clear or blue plaques, respectively. M13mp18
and M13mp19 differ with respect to the order of restriction sites within
the MCR.
Cosmids
Cosmids originate from the plasmid pBR322, but are inserted into cells by
transfection. To understand their function, we have to focus once again on
the replication of Iambda phage (Figure 4).
It is a typical feature ofA.-replication, that the phage DNAs areformedas one
long, so-called concatemeric molecule. Multiplegenomesare joined endto-end, separated by cohesive sites ( cos sites ). Cos sites are single-stranded,
complementary regions that are recognized by the packaging enzymes. The
DNA between cos sites is packaged into a phage particle as long as the distance between the two sites is in the range of 35-45 kb. This mechanism
provides the functional basis for cosmids (cos-site; plas-mid) (Collins
1979). pBR322 was changed so that a cos site was inserted into the tetracycline-resistance gene. Linearized plasmids will form concatameres similiar to those described for A.-replication. These cosmids arenot infectious,
since they Iack the phage's own coding sequences. However, in the presence
of enzymes ("packaging extract") and Iambda particles, in vitro packaging
of the recombinant DNA is efficient as long as the insert does not exceed[K.D.6] 35-45 kb in length. Cosmids replicate in the cell via the origin
of replication of pBR322 and may be isolated using the techniques described
for plasmids. Cosmid vectors are useful for genomic library construction
(Meyerowitz 1980).
Host cells
All ofthe host cells (Bachmann 1972, Bachmann 1980, Sambrook 1989) described below are derived from E. coli. Most of the vectors require specific
bacterial strains. For example, the use ofvectors expressing ß-galactosidase
gene activity requires the use oflac-, pro- bacterial strains, such as JM 103 ( see
M13 derivaties). A modern bacterial strain fulfilling this task is XL1-blue™
(Stratagene). Furthermore this strain can be grown on complete medium,
forms sex-pili and carries tetracycline-resistance-gene.