Biological Containment

A method of containing an extrachromosomal recombinant replicon to a first kind of bacterial cells, where said replicon could be naturally transferred to a second kind of bacterial cells, which comprises providing on the recombinant extrachromosomal replicon a first gene, expressed under the control of a regulatable promoter which is functional in both kinds of cells, whose expression results in formation of a toxic product which has a lethal effect on the first and second kind of cells, said first kind of cells having or being modified to have a second replicon comprising a second gene which encodes a gene product which inhibits the expression of said first gene and thereby protects said first kind of cells, said second gene being lacking in said second kind of cells, whereby if a cell of the second kind receives said extrachromosomal recombinant replicon said first gene is expressed and said toxic product is formed, which has a toxic effect thereon said first and second kinds of cells both being selected from the group consisting of Enterobacteriaceae, Pseudomonadaceae, and Bacillaceae cells, where said first gene comprises a coding sequence which encodes a polypeptide having an amino acid sequence wherein at least about 60% of the residues are identical to or conservative modifications of the corresponding residues of the amino acid sequence of the R1 Hok and/or F Hok polypeptides and/or the polypeptide encoded by relB-orf3.

A method of biologically containing bacterial cells growing in a first, controllable environment, which cells could escape to a second and physically distinct environment, which comprises:

• a) providing in said cell a recombinant replicon, said replicon comprising a first gene, expressed under the control of a regulatable promoter which is functional in said cells, whose expression results in formation of a toxic product which has a toxic effect on said cells, said cells natively containing or being modified to contain a second gene whose product inhibits expression of said first gene, said inhibition being regulatable by an environmental factor, the level of said environmental factor in the second environment being such that said first gene is expressed and said toxic product is formed, whereby cells in the second environment which harbor said replicon are killed, and
• b) manipulating the first environment such that the level of the environmental factor therein is such that expression of said first gene is inhibited, whereby cells bearing said replicon are able to grow in said first environment but not in said second environment, the toxic effect being such that at least 99.9% of the cells are killed in said second environment, which but for said effect is an environment in which said cells can grow where said bacterial cells are selected from the group consisting of Enterobacteriaceae, Pseudomonadaceae, and Bacillaceae cells, where said first gene comprises a coding sequence which encodes a polypeptide having an amino acid sequence wherein at least about 60% of the residues are identical to or conservative modifications of the corresponding residues of the amino acid sequence of the R1 Hok and/or F Hok polypeptides and/or the polypeptide encoded by relB-orf3.

A method of biologically containing bacterial cells growing in an initial environment subject to physical or chemical change resulting in a changed environment, so that said cells are able to grow in the initial environment but not in the changed environment, which comprises providing in said cells a recombinant replicon, said replicon comprising a first gene, expressed under the control of a regulatable promoter which is functional in said cells, whose expression results in formation of a toxic product which has a toxic effect on said cells, said cells natively containing or being modified to contain an inhibitory gene whose product inhibits expression of said first gene, said inhibition being regulatable by an environmental factor, the level of said environmental factor in said changed environment being such that said first gene is expressed and said toxic product is formed, whereby said cells in said changed environment which harbor said replicon are killed, the level of environmental factor in said initial environment being such that expression of said first gene is inhibited, whereby cells bearing said replicon are able to grow in the initial environment but not in the changed environment, the toxic effect being such that at least 99.9% of the cells are killed in said changed environment, which but for said effect is an environment in which said cells can grow, said cells being selected from the group consisting of Entero-bacteriaceae, Pseudomonadaceae, and Bacillaceae cells, where said first gene comprises a coding sequence which encodes a polypeptide having an amino acid sequence wherein at least about 60% of the residues are identical to or conservative modifications of the corresponding residues of the amino acid sequence of the R1 Hok and/or F Hok polypeptides and/or the polypeptide encoded by relB-orf3.