27916 
NOTICES 
when an infectious X containing cloned 
DNA infects a \-sensitive cell in nature, 
and recombines with a resident lambdoid 
prophage. Although X-sensitive E. coli 
strains seem to be rare, a significant frac- 
tion do carry lambdoid prophages (43- 
44) and thus this route of escape should 
be considered. 
While not exact, the estimates for 
containment afforded by using these 
host-vectors are at least as accurate as 
those for physical containment, and are 
sufficient to indicate that currently 
employed plasmid and X vector systems 
provide a moderate level of biological 
containment. Other nonconjugative plas- 
mids and bacteriophages that, in asso- 
ciation with E. coli K-12 can be estimated 
to provide the same approximate level of 
moderate containment are included in 
the EK1 class. 
EK2 host-vectors. These are host-vec- 
tor systems that have been genetically 
constructed and shown to provide a high 
level of biological containment as demon- 
strated by data from suitable tests per- 
formed in the laboratory. The genetic 
modifications of the E. coli K-12 host 
and/or the plasmid or phage vector 
should not permit survival of a genetic 
marker carried on the vector, preferably 
a marker within an inserted DNA frag- 
ment, in other than specially designed 
and carefully regulated laboratory en- 
vironments at a frequency greater than 
10“*. This measure of biological contain- 
ment has been selected because it is a 
measurable entity. Indeed, by testing the 
contributions of preexisting and newly 
introduced genetic properties of vectors 
and hosts, individually or in various com- 
binations, it should be possible to esti- 
mate with considerable precision, that 
the specially designed host-vector system 
can provide a margin of biological con- 
tainment in excess of that required. For 
the time being, no host- vector system will 
be considered to be a bona fide EK2 host- 
vector system until it is so certified by the 
NTH Recombinant DNA Molecule Pro- 
gram Advisory Committee. 
For EK2 host-vector systems in which 
the vector is a plasmid, no more than one 
in 10 s host cells should be able to per- 
petuate the vector and/or a cloned DNA 
fragment under non-permissive condi- 
tions designed to represent the natural 
environment either by survival of the 
original host or as a consequence of 
transmission of the vector and/or a 
cloned DNA fragment by transformation, 
transduction or conjugation to a host 
with properties common to those in the 
natural environment. 
In terms of potential EK2 plasmid-host 
systems, the following types of genetic 
modifications should reduce survival of 
cloned DNA. The examples given are for 
illustrative purposes and should not be 
construed to encompass all possibilities. 
The presence of the non-conjugqtive 
plasmids ColEl-frp and pSIOl in an 
E. coli K-12 strain possessing a mutation 
eliminating host-controlled restriction 
and modification ( hsdS ) results in about 
lOMold reduction in mobilization to re- 
striction-proficient recipients. The com- 
bination of the dapD8, AbioH-asd, Agal- 
chl r and rfb mutations in E. coli K-12 
results in no detectable survivors in feces 
of rats following feeding by stomach tube 
of 10“ cells in milk and similarly leads to 
complete lysis of cells suspended in broth 
medium lacking diaminopimelic acid. 
E. coli K-12 strains with AthyA and deoC 
(dr a) mutations undergo thymineless 
death in growth medium lacking thymine 
and give a 10 r -fold reduced survival dur- 
ing passage through the rat intestine 
compared to wild-type thy * E. coli K-12. 
(However, the A thy A mutation alone or 
in combination with a deoB(drm) muta- 
tion only reduces in vivo survival by a 
factor of 10 2 .) Other host mutations, as 
yet untested, that might further reduce 
survival of the plasmid-host system or 
reduce plasmid transmission are : the 
combination poZA(TS) recA(TS) AthyA 
which might interfere with ColEl repli- 
cation and lead to DNA degradation at 
body temperatures; Con - mutations that 
reduce the ability of conjugative plasmids 
to enter the plasmid-host complex and 
thus should reduce mobilization of the 
cloned DNA to other strains; and muta- 
tions that confer resistance to known 
transducing phages. Mutations can also 
be introduced into the plasmid to cause 
it to be dependent on a specific host, to 
make its replication thermosensitive 
and/or to endow it with a killer capa- 
bility 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, prefer- 
ably deletions. Obviously, the presence of 
all mutations contributing to higher de- 
grees of biological containment must be 
verified periodically by appropriate tests. 
In testing the level of biological contain- 
ment afforded by a proposed EK2 plas- 
mid-host system, it is important to de- 
sign relevant tests to evaluate the sur- 
vival of the vector and/or a cloned DNA 
fragment under conditions that are pos- 
sible in nature and that are also most 
advantageous for its perpetuation. For 
example, one might conduct a triparental 
mating with a primary donor possessing 
a derepressed F-type or I-type conjuga- 
tive plasmid, the safer host with AbioH- 
asd, dapD8, Agal-chl T , rfb, AthyA, deoC, 
trp and hsdS mutations and a plasmid 
vector carrying an easily detectable 
marker such as for ampicillin resistance 
or an inserted gene such as trp*, and a 
secondary recipient that is Su + hsdS trp 
(i.e., permissive for the recombinant 
plasmid) . Such matings would be con- 
ducted in a'medium lacking diaminopi- 
melic acid and thymine and survival 
of the Ap 1, or trp * marker in any of the 
three strains followed as a function of 
time. Survival of the vector and/or a 
cloned marker by transduction could also 
be evaluated by introducing a known 
generalized transducing phage into the 
system. Similar experiments should also 
be done using a secondary recipient that 
is restrictive for the plasmid vector as 
well as with primary donors possessing 
repressed conjugative plasmids with in- 
compatibility group properties like those 
commonly found in enteric microorgan- 
isms. Since a common route of escape 
of plasmid-host systems in the labora- 
tory might be by accidental ingestion, it 
is suggested that the same types of ex- 
periments be conducted in suitable ani- 
mal-model systems. In addition to these 
tests on survival of the vector and/or a 
cloned DNA fragment, it would be useful 
to determine the survival of the host 
strain under nongrowth conditions such 
as in water and as a function of drying 
time after a culture has been spilled on 
a lab bench. 
For EK2 host-vector systems in which 
the vector is a phage, no more than one 
in 10 8 phage particles should be able to 
perpetuate itself and/or a cloned DNA 
fragment under non-permissive condi- 
tions designed to represent the natural 
environment either (a) as a prophage 
or plasmid in the laboratory host used for 
phage propagation or (b) by surviving 
in natural environments and transfer- 
ring itself and/or a cloned DNA frag- 
ment to a host (or its resident lamboid 
prophage* with properties common to 
those in the natural environment. 
In terms of potential EK2 x-host sys- 
tems, the following types of genetic modi- 
fication should reduce survival of cloned 
DNA. The examples given are for illus- 
trative purposes and should not be con- 
strued to encompass all possibilities. The 
probability of establishing X lysogeny in 
the normal laboratory host should be re- f 
duced by removal of the phage att site, 
the Int function, the repressor gene(s) I 1 
and adding virulence-enhancing muta- 
tions. The frequency of plasmid forma- 1 
tion, although normally already less than 
10 A could be further reduced by defects 
in the p R -Q region, including mutations 
such as vir-s, cro( TS), ci7 t ri', O(TS), 
P(TS), and nin. Moreover, chloroform n 
treatment used routinely following cell 
lysis would reduce the number of surviv- *i 
ing cells, including possible lysogens or H 
plasmid carriers, by more than 10 8 . The 
host may also be modified by deletion 
of the host xatt site and inclusion of one 
or more of the mutations described above 
for plasmid-host systems to further re- 
duce the chance of formation and sur- 
vival of any lysogen or plasmid carrier 
cell. 
The survival of escaping phage and the 
chance of encountering a sensitive host 
in nature are very low, as discussed for 
EK1 systems. The infectivity of the phage 
particles could be further reduced by in- jj 
troducing mutations (e.g., suppressed |; 
ambers) which would make the phage !> 
particles extremely unstable except un- 
der special laboratory conditions (e.g_ -j 
high concentrations of salts or putres- 
cine) . Another means would be to make hi 
the phage itself a two-component system, [J 
by eliminating the tail genes and re- 
producing the phage as heads packed 
with DNA; when necessary and under 
specially controlled conditions, these 
heads could be made infective by adding i 
tail preparations. An additional safety 
factor in this regimen is the extreme 
instability of the heads, unless they are 
stored in lOmM putrescine, a condition 
easy to obtain in the laboratory but not 
in nature. The propagation of the es- 
FEDERAL REGISTER, VOL. 41, NO. 131 — WEDNESDAY, JULY 7, 1976 
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