15 Cloning Vectors
227
• The transcription of isolated genes into corresponding mRNA as a first
step of in vivo or in vitro synthesis of their encoded proteins
• The possibility of further subcloning
• A high rate of biological securityModern vectors are designed by DNA
engineering to fit a certain range of the demands listed above. W e will
focus on typical sequencing vectors (M13) as well as typical expression
vectors.
Principles of cloning
The steps of cloning are shown in Figure 1 using a plasmid. First, the circular
plasmid is linearized using a restriction endonuclease that cuts at a unique
recognition sequence producing a single cleavage. The sticky ends or blunt
ends formed are characteristic for each of the restriction enzymes ( see Chap.
2). The DNA inserttobe cloned into the vector is produced by digestion with
the same restriction enzyme or an isoschizomer to produce compatible
ends. Ligation is performed by joining the ends of foreign DNA and linearized plasmid using a ligase (see Chap. 2), resulting in a circular chimerk
plasmid. Ligation is followed by transformation of bacterial host cells
(when using bacteriophage, this step is called transfection). The bacterial
host cell is made competent' to receive the plasmid through alteration of the
bacterial cell wall using CaClz or an electrical im pulse. As a safety measure,
natural transformation oflaboratory plasmids is inhibited by removing the
genes coding for this ability. Once inside the cell, replication of the plasmid
is initiated from an origin of replication ( ori). U sing multicopy plasmids, all
cells originating from the transformed cell will bear several copies of the
recombinant plasmid. Following in vitro celllysis, these plasmids can be
isolated and the insert, now present in several thousands of identical copies,
can be excised, usually using the same restriction enzymes as for initial
ligation. Some phages will cause Iysis of the transfected cells (temperate
phage). These can be isolated from the supernatant after the lytic cycle
has been completed.
Plasmids
Plasmids are small, circular, double-stranded DNA elements (Figure 2).
They replicate autonomously in bacterial cells independently of the bacterial chromosome. They are known to the physician as transmitters of bacterial antibiotic resistence. Plasmid vectors are composed of:
227
• The transcription of isolated genes into corresponding mRNA as a first
step of in vivo or in vitro synthesis of their encoded proteins
• The possibility of further subcloning
• A high rate of biological securityModern vectors are designed by DNA
engineering to fit a certain range of the demands listed above. W e will
focus on typical sequencing vectors (M13) as well as typical expression
vectors.
Principles of cloning
The steps of cloning are shown in Figure 1 using a plasmid. First, the circular
plasmid is linearized using a restriction endonuclease that cuts at a unique
recognition sequence producing a single cleavage. The sticky ends or blunt
ends formed are characteristic for each of the restriction enzymes ( see Chap.
2). The DNA inserttobe cloned into the vector is produced by digestion with
the same restriction enzyme or an isoschizomer to produce compatible
ends. Ligation is performed by joining the ends of foreign DNA and linearized plasmid using a ligase (see Chap. 2), resulting in a circular chimerk
plasmid. Ligation is followed by transformation of bacterial host cells
(when using bacteriophage, this step is called transfection). The bacterial
host cell is made competent' to receive the plasmid through alteration of the
bacterial cell wall using CaClz or an electrical im pulse. As a safety measure,
natural transformation oflaboratory plasmids is inhibited by removing the
genes coding for this ability. Once inside the cell, replication of the plasmid
is initiated from an origin of replication ( ori). U sing multicopy plasmids, all
cells originating from the transformed cell will bear several copies of the
recombinant plasmid. Following in vitro celllysis, these plasmids can be
isolated and the insert, now present in several thousands of identical copies,
can be excised, usually using the same restriction enzymes as for initial
ligation. Some phages will cause Iysis of the transfected cells (temperate
phage). These can be isolated from the supernatant after the lytic cycle
has been completed.
Plasmids
Plasmids are small, circular, double-stranded DNA elements (Figure 2).
They replicate autonomously in bacterial cells independently of the bacterial chromosome. They are known to the physician as transmitters of bacterial antibiotic resistence. Plasmid vectors are composed of: