20 
Mutations oan also be introduced into the -plasmid to cause it 
to be dependent on a specific host 3 to make its replication 
thermo sensitive and/or to endcw it with a killer capability such 
that all cells (other than its host) into which it might be 
transferred will not survive. 
In the construction of EK2 plasmid-host systems it is 
important to use the most stable mutations available 3 preferably 
deletions. Obviously 3 the presence of all mutations contributing 
to higher degrees of biological containment must be verified 
periodically by appropriate tests. In testing the level of 
biological containment afforded by a proposed EK2 plasmid-host 
system 3 it is important to desig'n relevant tests to evaluate 
the survival of the cloned DNA under conditions that are possible 
in nature and that are also most advantageous for its perpetuation. 
For example 3 one might conduct a triparental mating with a primary 
donor possessing a derepressed F-type or I-type con/ugative plasmid 3 
the safer host with A bioH-asd 3 dapD8 3 A gal-chl 3 A thy A 3 deoC 3 trp and 
hsdS mutations and a plasmid vector carrying an easily detectable 
inserted gene such as for ampicillin resistance or trp + 3 and a 
secondary recipient that is Su + hsdS trp (i.e. 3 permissive for the 
recombinant plasmid ) . Such matings would be conducted in a medium 
lacking diaminopimelic acid and thymine and survival of the A.p r 
or trp + marker in any of the three strains followed as a function 
of time. Survival of the cloned marker by transduction could also 
be evaluated by introducing a known generalized transducing phage 
[ 91 ] 
