Screening for Productive Strains and Strain Improvement 143
antibiotics, and unusual metabolites. In some cases they may carry genes for unusual
capabilities such as the breakdown of complex organic compounds. Plasmids are,
however, not essential for the cell’s survival.
Two important features of plasmids to be used in genetic experiments may be
compared by examining two plasmids. Plasmid psC101 has only two to five copies per
cell and replicates with its host DNA. It is said to be under ‘stringent’ control. However,
another plasmid pCol E 1 is found in about 25–30 copies per cell. It has a ‘relaxed control’
independent of the host and replicates without reference to the host DNA. When the host
cell is starved of amino-acids or its protein synthesis is inhibited in some other manner,
such as with the use of chloramphenicol, the Col E 1 plasmid continues to replicate for
several hours until there are 1,000 to 3,000 copies per cell. Due to this high level of gene
dosage (also referred to as gene amplification), products synthesized because of the
presence of these plasmids are produced in extremely high amounts, a property of
immense importance in biotechnology and industrial microbiology. Generally
conjugative plasmids are large, exhibit stringent control of DNA replication, and are
present in low copy numbers; on the other hand, non-conjugative plasmids are small,
show relaxed DNA replication, and are present in high numbers. Many other plasmid
vectors exist, some constructed in the laboratory (Table 7.5).
(i) Ideal properties in a plasmid used as a vector
A plasmid to be used in genetic engineering should ideally have the following properties:
(a) the plasmid should be as small as possible so the unwanted genes are not
transmitted, as well as to facilitate handling;
(b) it should have an origin of replication, the site where DNA replication initiates;
(c) it should have a relaxed mode of replication;
(d) it should have sites for several restriction enzymes;
(e) it should carry, preferably, two marker genes. Marker genes are those which
express characteristics by which the plasmid can be identified. Such
characteristics include resistance to one or more antibiotics. A marker of great
importance is the ability to satisfy auxotrophy, i.e., the ability to produce an amino
acid or other nutritional component which the host’s chromosome is incapable of
producing.
Table 7.5 Some commonly used plasmid cloning vehicles
Plasmid Molecular weight Marker* Single restriction sites
(x 10
-6
)
pSC101 5.8 Tc BamHI, EcoRI, HindIII, Hpal, SalI, Smal
Col E1 4.2 Colimm EcoRI
pMB9 3.6 Tc
r
, Colimm BamHI, EcoRI, HindIII, Hpal, SalI Smal
pBR313 5.8 Tc
r
, Ap
r
BamHI, EcorI, HindIII, Hpal, SalI, Smal
Colimm
pBR322 2.6 BamHI, EcoRI, HindIII, pstI, SalI
*Tc
r
: tetracycline resistance. Ap
r
: ampicillin resistance
colimm: colicin immunity