5 
Dr. Gottesman asked how much Shiga toxin would be produced in the human gut in 
a worst case scenario in which all of the EL_ coli in the gut (assumed to be 
approximately 10^ EL_ coli ) were expressing Shiga toxin at theoretical maximum 
possible levels. She asked whether this amount of toxin would cause disfigure- 
ment and death. 
Dr. Formal asked if the amount of toxin produced in the gut was a consideration 
when the proposal to clone the cholera toxin gene was evaluated. Dr. Gill 
said it was not a concern with cholera toxin; if more cholera toxin is introduced 
into the gut, no symptoms other than diarrhea occur. He thought the amount of 
toxin which could potentially be produced should be evaluated, however, if the 
Shiga toxin gene is cloned on a multicopy plasmid. He pointed out that Shiga 
toxin is cytotoxic and could have effects other than diarrhea. 
Dr. Gottesman calculated that in a worst case scenario, 10 9 engineered Eh_ coli 
with the Shiga toxin gene on a high expression, high copy number plasmid might 
produce one milligram of toxin in the human gut. Dr. Gill calculated that this 
amount is roughly equivalent to approximately 14,000 lethal doses for humans 
when the toxin is administered parenterally. He said that 100,000 times more 
tetanus toxin is required enterally to kill animals than is required parenterally. 
Dr. Gottesman noted that Branham, Dack, and Riggs had administered 20,000 lethal 
doses to monkey intestinal pouches with no observed effect (Attachment III). 
Dr. Levine moved that the working group recommend to RAC that cloning of the 
Shiga toxin gene in coli be permitted at P3 containment. Dr. Gill seconded 
the motion. By a vote of five in favor, none apposed, and no abstentions, the 
working group accepted the motion. 
Dr. Gottesman asked if Dr. O'Brien's proposal requests permission to use EKl 
phage vectors as well as EKl plasmid vectors. Dr. Holmes said the proposal 
referred to EKl phage vectors as well as EKl plasmid vectors. 
Dr. Gottesman felt resolution of the issues involved in the third item of 
Dr. O'Brien's request might aid in reaching a consensus on the second item. 
She, therefore, suggested the working group next consider the third item of the 
proposal which reads as follows: 
"3. If we can identify nontoxinogenic fragments of the structural gene(s) 
for Shiga-like toxin, we request permission to: a) remove any such 
cloned nontoxic fragments (generated during the search for clones that 
contain intact toxin structural genes) from the original containment 
conditions to work with them under PI + EKl conditions; b) directly 
clone any such nontoxic fragments into E. coli K-12 under Pi conditions." 
Dr. Gill felt this item implicitly referred to cloning under conditions in 
which the fragments of the gene encoding the toxin will not have the opportunity 
to recombine to regenerate the complete Shiga toxin structural gene. Dr. O'Brien 
agreed that it was, but noted that the arrangement of the Shiga toxin structural 
gene is unknown. 
[414] 
