vectors

VECTORS

Both prokaryotic and eukaryotic host-vector systems can be used in GM work. This section initially concentrates on prokaryotic host-vector systems with some details of eukaryotic ones being given later.

A prokaryotic vector should:

1. Be capable of autonomous replication independent of the main bacterial chromosome, i.e. possess an origin of replication (ori).

2. Be easy to isolate, i.e. small.

3. Be non-toxic to host cells.

4. Have space for foreign inserts.

5. Have unique restriction sites for common restriction enzymes.

6. Have convenient markers for selection of transformants, e.g. antibiotic resistance genes.

7. Be relaxed, i.e. multiple copies in a host cell.

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Most prokaryotic vectors are based on :

1. Plasmids

2. Bacteriophages

3. Cosmids (artificial constructions)

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1. PLASMID VECTORS

Plasmids are:

Circular, autonomous molecules of DNA.

Found naturally in most bacterial (and some other) species.

Size: 1.5 - 300 kilobases.

Function: carry non-essential (dispensable) genes, e.g. antibiotic resistance, toxin production.

But "cryptic" plasmids have no known function!

Plasmids can be conjugative or non-conjugative (conjugation is generally not required in GM).

Plasmids can be mobilizable or non-mobilizable (non-mobilizable plasmids are preferred as they are less likely to "escape" from host cells).

Plasmids can be relaxed (multiple copies per host cell) or stringent (1-3 copies per host cell).

For GM work we want: small, relaxed, non-conjugative, non-mobilizable plasmids with good markers and unique restriction sites.

pBR322-An example of an artificial plasmid cloning vector.