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Appendix F-IV-F. The gene(s) coding 
for a toxin (designated LT-like) isolated 
from E. coli which is similar to the E. 
coli beat labile enterotoxin (LT) with 
respect to its activities and mode of 
action but is not neutralized by 
antibodies against cholera enterotoxin 
or against LT from human cr porcine E. 
coli strains, and sequences homologous 
to the E. coli LT-like toxin ger.e may be 
cloned under BLl+EXl conditions. 
Appendix F-IV-G. Genes from Vibrio 
fluvialis. Vibrio mimicus. and non 0-1 
Vibrio chclerae. specifying virulence 
factors for animals, may be cloned 
under ELl + EKl conditions. The 
virulence factors to be cloned will be 
selected by testing fluid induction in 
suckling mice and Y-l mouse adrenal 
cells. 
Appendix F-I\'-H. The intact 
structural gene(s) of the Shiga-like toxin 
from bacterial species classified in the 
families Enterobcctericceae or 
Vibrionaceae including Campylobacter 
species may be cloned in E. coli K-12 
under BL3+EK1 containment 
conditions. 
E. coli host-vector systems expressing 
the Shiga-like toxin gene product may 
be moved from BL3+EK1 to BL2 + EK1 
containment conditions provided that: 
(1) The amount of toxin produced by the 
modified host-vector systems be no 
greater than that produced by the 
positive control strain Shigella 
dysenteriae 6GR. grown and measured 
under optimal conditions: and (2) the 
cloning vehicle is to be an EXl vector 
preferably belonging to the class of 
poorly mobilizable plasmids such as 
pER322, pBk328, and pBR325. 
Nontoxinogenic fragments of the 
Shiga-like toxin structural gene(s) may 
be moved from BL3 + EK1 to BL2-j-EKl 
containment conditions or such nontoxic 
fragments may be directly cloned in E. 
coli K-12 under BL2 + EK1 conditions 
provided that the E. coli host-vector 
systems containing the fragments do not 
contain overlapping fragments which 
together would encompass the Shigalike 
toxin structural gene(s). 
Appendix F-IV-l. A hybrid gene in 
which the gene coding for the 
melanocyte stimulating hormone (MSH) 
is joined to a segment of the gene 
encoding diphtheria toxin may be safely 
propagated in E. coli K-12 under BL4 
containment in high containment 
building 550 at the Frederick Cancer 
Research Facility. If the investigators 
wish to proceed with the experiment, a 
prior review will be conducted to advise 
NTH whether the proposal has sufficient 
scientific merit to justify the use of the 
NTH BL4 facility. Before any of the 
strains may be removed from the BL4 
facility, data on their safety shall be 
[ 16 ] 
evaluated by the Working Group in 
Toxins and the working group 
recommendation shall be acted upon by 
NIH. 
Appendix F-IV-J. The gene segment 
encoding the A subunit of chlolera toxin 
of Vibrio cholerae may be joined to the 
transposons Tn5 and Tn5-131 and the 
A-subunit::Tn5-131 hybrid gene cloned 
in E. coli K-12 and V. cholerae under 
BLl containment Conditions. 
Appendix F-IV-K. A hybrid gene in 
which the gene coding for interleukin 2 
(1L-2) is joined to a specific segment of 
the gene encoding diphtheria toxin may 
be propagated in E. coli K-12 host- 
vector systems under BL2 containment 
plus BL3 practices, with the use of 
poorly mobilizable plasmid vectors such 
as EK2 certified plasmids. 
Appendix G — Physical Containment 
Appendix G-l — Standard Practices and 
Training 
The first principle of containment is a 
strict adherence to good microbiological 
practices [1-10]. Consequently, all 
personnel directly or indirectly involved 
ir. experiments on recombinant DMAs 
must receive adequate instruction (see 
Sections IV-B-l-e and IV-B-5-d). This 
shall, as a minimum, include instructions 
in aseptic techniques and in the biology 
of the organisms used in the 
experiments so that the potential 
biohazards can be understood and 
appreciated. 
Any research group working with 
agents with a known or potential 
biohazard shall have an emergency plan 
which describes the procedures to be 
followed if an accident contaminates 
personnel or the environment. The PI 
must ensure that everyone in the 
laboratory is familiar with both the 
potential hazards of the work and the 
emergency plan (see Sections IV-B-3-d 
and IV-B-5-e). If a research group is 
working with a known pathogen for 
which there is an effective vaccine, the 
vaccine should be made available to all 
workers. Where serological monitoring 
is clearly appropriate, it shall be 
provided (see Section IV-B-l-f). 
The “Laboratory’ Safety Monograph" 
and Biosafety in Microbiological and 
Biomedical Laboratories [2] booklets 
describe practices, equipment, and 
facilities in detail. 
Appendix G-Il — Physical Containment 
Levels 
The objective of physical containment 
is to confine organisms containing 
recombinant DMA molecules and thus to 
reduce the potential for exposure of the 
laboratory worker, persons outside of 
the laboratory, and the environment to 
organisms containing recombinant DNA 
molecules. Physical containment is 
achieved through the use of laboratory 
practices, containment equipment, and 
special laboratory design. Emphasis is 
placed on primary means of physical 
containment which are provided by 
laboratory practices and containment 
equipment. Special laboratory design 
provides a secondary means of 
protection against the accidental release 
of organisms outside the laboratory or to 
the environment. Special laboratory 
design is used primarily in facilities in 
which experiments of moderate ic high 
potential hazards are performed. 
Combinations of laboratory’ practices, 
containment equipment, and special 
laboratory design can be made to 
achieve different levels of physical 
containment. Four levels of physical 
containment, which are designated as 
BLl. BL2, BL3, and BLl. are described. It 
should be emphasized that the 
descriptions and assignments of 
physical containment detailed below are 
based on existing approaches to 
containment of pathogenic organisms 
[2]. The National Cancer Institute 
describes three levels for research on 
oncogenic viruses which roughly 
correspond to our BI.2, BL3. and BLl 
level [3]. 
It is recognized that several different 
combinations of laboratory’ practices, 
containment equipment, and special 
laboratory design may be appropriate 
for containment of specific research 
activities. The Guidelines, therefore, 
allow alternative selections of primary 
containment equipment within facilities 
that have been designed to provide EL3 
and BLl levels of physical containment. 
The selection of alternative methods of 
primary’ containment is dependent, 
however, on the level of biological 
containment provided by the host-vector 
system used in the experiment. 
Consideration will also be given by the 
Director, NIH, with tha advice of the 
RA.C to other combinations which 
achieve an equivalent level of 
containment (see Section IV-C-l-b-{2)- 
(bJJ. 
Appendix C-II-A — Biosafety Level 1 
(BLl) [13] 
Appendix G-II-A-1. Standard 
Micro biological Practices. 
Appendix G-II-A-l-a. Access to the 
laboratory is limited or restricted at the 
discretion of the laboratory director 
when experiments are in progress. 
Appendix C-II-A-l-b. Work surfaces 
are decontaminated once a day and 
after any spill of viable material. 
Recombinant DNA Research, Volume 1 1 
