APPENDIX E 
containment for experiments with E. col i systems. Other prokaryote host- 
vector systems need to be evaluated using the same general principles as 
those appi ied to the E. col i K12 situation. 
As with physical containment levels, increasing numbers specify increasing 
levels of biological containment for E. col i systems. The next level is called 
EK2. EK2 host-vector combinations must be demonstrated to provide a high level 
of biological containment by suitable laboratory tests. They are obtained by 
genetic modification of either E. co I i K12 host cel Is or the relevant plasmids 
and bacteriophage or both. More specifically, the guidelines state that in 
order to qualify as EK2 the modified system composed of derivatives of E. col i 
K12 combined with a particular vector should not permit survival of a genetic 
marker carried on the vector in other than specially designed I aboratory environ 
-8 
ments at a frequency greater than 10 . Various examples of the types of 
necessary modifications are suggested in the guidelines. For example, modifi- 
cations of the host might be mutations which result in special nutritional 
requirements for growth or sensitivity to naturally occuring materials such as 
bile salts, or elimination of host-controlled restriction and modification. 
Suggested modifications of plasmid vectors include mutations making essential 
plasmid functions sensitive to normal body temperatures or dependent on a specif 
host. Mutations which make native phage particles containing a recombinant DNA 
unstable in natural environments and therefore unlikely to infect new E. col i 
cells should they escape can be considered. 
One additional level of contained E. col i host-vector systems is defined 
in the guidelines and is called EK3. EK3 systems are EK2 systems for which the 
specified containment properties have been demonstrated not only by micro- 
biological and genetic anaylsis but by appropriate tests in animals including 
Appendix E — 12 
