APPENDIX C 
Summary of the Workshop on the DESIGN AND TESTING OF 
SAFER PROKARYOTIC VEHICLES AND BACTERIAL HOSTS FOR 
RESEARCH ON RECOMBINANT DNA MOLECULES 
Torrey Pines Inn, La Jolla, California 
The development of techniques for the cloning of DNA from both prokaryotic and 
eukaryotic organisms in bacteria has had great impact on research in biology and 
medicine and promises extraordinary social benefits. The biohazards involved in the 
use of this technology in many instances is very difficult to assess. For this 
reason codes of practice are being formulated in the United States and other coun- 1 
tries for the conduct of those experiments that present a potential biohazard. One j 
of the requirements for conducting certain cloning experiments is the use of safer 
vector (bacteriophage or plasmid) -host systems, i.e., vector-bacterium systems 
that have restricted capacity to survive outside of controlled conditions in the 
laboratory. Approximately sixty scientists from the United States and several 
foreign countries participated in a workshop on the Design and Testing of Safer 
Prokaryotic Vehicles and Bacterial Hosts for Research on Recombinant DNA Molecules 
at La Jolla, California, on 1 to 3 December, 1975. The workshop was sponsored by 
the Research Resources Branch of the National Institute of Allergy and Infectious 
Diseases. The purposes of the meeting were the exchange of recent data on the 
development of safer prokaryotic host-vector systems, devising methods of testing 
the level of containment provided by these systems and exploring the various direc- 
tions that future research should take in the construction of safer bacterial 
systems for the cloning of foreign DNA. 
The first session of the workshop, chaired by W. Szybalski (University of 
Wisconsin), was devoted to bacteriophage vectors. Szybalski outlined the main 
safety features of the two-ccmponent, phage-bacterial system, in which the host 
bacteria offer the safety feature of not carrying the cloned DNA, and the phage 
vectors cannot be propogated in the absence of an appropriate host. There are two 
primary escape routes for the clones of foreign DNA carried by the phage vector: 
(1) establishment of a stable prophage or plasmid in the laboratory host used for j 
phage propagation, and subsequent escape of this self replicating lysogen or carrier 
system, and (2) escape of the phage vector which carries the cloned DNA and its 
subsequent productive encounter with a suitable host in the natural environment. 
The general concensus was that to ensure safety, both routes should be blocked by 
appropriate genetic modifications. For phage X, route (1) can be blocked by phage 
mutations that interfere with lysogenization (att ~ , int~ cl", cIII", viv) and 
plasmid formation (AT*", ninR, VS , ri c , oil, Ots , cvo ts) , and by mutations on the 
Escherichia colt host that affect these processes (at£B~, dncAts) and host survival.! 
Route (2) , [which is of low probability since A phages do not survive well in 
natural environments (no Acl phage was recovered after ingestion of 1C)8-10^ par- 
ticles) , are killed by desiccation, and have a low chance to encounter a naturally 
sensitive host] can be blocked further by the following phage modifications: (a) 
mutations which result in extreme instability of the infectious phage particles 
under all conditions other than those specially designed for phage propagation in 
the laboratory (e.g., high concentrations of putrescine or some other compound), or 
(b) employing phage vectors in which the tail genes are deleted and which permit 
propagation, of only the DNA-packed heads; only under laboratory conditions could 
such heads be made transiently infectious by t e joining them with separately pre- 
pared tails. The high instability of the phage would minimize the possibility of 
[ 134 ] 
