Evolution and Genetic Engineering - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 715

A ─A ─T ─T ─C ─A ─T ─G ─

+

+

─ A ─ C ─ T ─ G ─A ─C ─T ─G A ─A ─T ─T ─C ─A ─T ─G ─

G ─T ─A ─C ─

─ T ─G ─A ─C ─T ─T ─A ─A ─T ─G ─A ─C ─T ─T ─A ─A G ─ T ─ A ─ C ─

Complimentary
base pairing

DNA ligase

─A ─C ─T ─G ─ A ─A ─T ─T ─C ─A ─T ─G ─

─T ─G ─A ─C ─T ─T ─A ─A ─ G ─ T ─ A ─ C ─

FIGURE 14.9 Attaching a foreign gene onto a DNA molecule (cut by restrictive enzyme).

Avector is needed because a length of DNA containing a gene on its own will not actually
do anything inside a host cell. Since it is not part of the cell's normal genome, it will not be
replicated when the cell divides, it will not be expressed, and in fact it will probably be
broken down pretty quickly. Avector gets around these problems by having these properties:
￿

It is big enough to hold the gene we want (plus a few others) but not too big.

￿ It is circular (or more accurately a closed loop), so that it is less likely to be broken down

(particularly in prokaryotic cells where DNA is always circular).

￿ It contains control sequences, such as a replication origin and a transcription promoter, so

that the gene will be replicated, expressed, or incorporated into the cell's normal genome.

￿ It contains marker genes, so that cells containing the vector can be identified.

Many different vectors have been made for different purposes in genetic engineering
by modifying naturally occurring DNA molecules, and these are now available off the
shelf. Different kinds of vector are also available for different lengths of DNA insertion
( Tabl e 14 . 4 ).

14.4.5.3. Plasmids
Plasmids are by far the most common kind of vector, so we shall look at how they are used
in detail. Plasmids are short circular bits of DNA found naturally in bacterial cells. A typical
plasmid contains three to five genes and there are usually around 10 copies of a plasmid in
a bacterial cell. Plasmids are copied separately from the main bacterial DNA when the cell

TABLE 14.4 Gene Insertion Length into DNAMolecule as a Function of the Vector

Type of vector Maximum length of DNA insertion

Plasmid 10 kbp

Virus of phage 30 kbp

Bacterial artificial chromosome (BAC) 500 kbp