Approaches for Monitoring and Containing Genetically Engineered Microorganisms Released into the Environment

Abstract

We have examined methods for tracking and limiting the survival capacity of genetically engineered microorganisms (GEMs) in the environment. By coupling efficient DNA extraction methodology with the poly–merase chain reaction (PCR) for the invitro amplification of a gene probe target, we were able to detect 1 cell of a target organism/g sample (100 organisms in a 100g soil or sediment sample) by dot-blot hybridization against a background of 1011 diverse organisms. This is at least three orders of magnitude better sensitivity than has been achieved by previous gene probe methods. We have also constructed a model conditional-lethal plasmid vector (pBAP19h) for containing GEMs in the environment by placing the host killing gene (hok) from pPR633 under the control of the lac promoter and operator of plasmid pTZ19u in E. coli. In invitro experiments, cultures inoculated with E. coli JM101 containing the conditional lethal ("suicide") plasmid pBAP19h ceased to grow 1-2 h after addition of IPTG, but returned to exponential growth after an additional 2 h of incubation; however, cells isolated 4 h after addition of IPTG no longer contained pBAP19h. When carbenicillin was added as a selective pressure for maintaining the plasmid, the suicide plasmid failed and after an initial period of cell decline, pBAP19h-bearing cells that had developed Hok resistance grew exponentially. In soil microcosm studies, the addition of IPTG resulted in a 90% decline in cells containing pBAP19h within 24 h of Hok induction and there was no evidence of renewed growth of hok-containing cells. Surviving cells, still containing hok were debilitated; they were not capable of normal growth, nor could they stably maintain the conditional-lethal plasmids. These results indicate that, although not yet a fail safe system, hok-bearing "suicide" vectors have the potential for limiting the spread of GEMs in the environment.