cells are viable only in the presence of 3-methylbenzoate. Cells not having
3-methylbenzoate would not survive, either because of its degradation or because
of its escape to the environment, because of Gef expression. The escape rate from
killing in the lab was approximately 10
À8 cell
À1 generation
À1 . The testing of the
system was done in fields with bulk soil or rhizosphere soil and was very efficient.
There was no proof that it spread out of the experimental area (Torres et al. 2003a).
To evade lethal function loss because of mutation, double strains with extra function
depending upon the asd gene were made for diaminopimelic acid synthesis. This
gene is compulsory needed in cell wall synthesis, and strains without it need totally
external diaminopimelic acid, which is not present in the soil environments. A
P. putida strain with asd gene deletions has an alternative asd gene which is under
positive regulation by the XylS activator. This same XylS activator negatively
regulates gef expression too through transcription of the lacI gene as explained
earlier. Therefore, a strain devoid of 3-methylbenzoate would not survive in an
environment because of killing by Gef protein and also because of the absence of
diaminopimelic acid. The survival level of this strain due to the lack of
3-methylbenzoate was under the detection range (<10
À9 cell
À1 generation
À1
(Ronchel and Ramos 2001).
12.12.6 Suicide Mechanisms Preventing Horizontal Gene Transfer
The suicide scheme is created to allow reliable killing of recombinant strains from
being discharged into the wild. The same type of schemes could stop the transfer of
recombinant genes into other bacteria through conjugation, transduction or transformation. One diverse conditional lethal scheme was dependent on the colicin E3
(colE3) gene which kills several diverse bacteria through breaking of 16S ribosomal
RNA (Torres et al. 2003b). Under natural condition, this lethal function is opposed
through an immunity function (encoded by the immE3 gene) providing a poison/
antidote effect. The colE3 gene was kept inside a plasmid, and the immE3 gene was
put on the chromosome of E. coli or P. putida to test whether the horizontal transfer
of plasmid DNA to other bacteria could be stopped by using the killing function.
Therefore, horizontal transfer of the plasmid to other bacteria would cause cell death
in the recipient because the colE3 gene would be transferred without chromosomal
immE3 gene. This scheme has the capability to stop horizontal transfer into a broad
range of Gram-negative bacteria (Torres et al. 2003a). The main trouble with this
kind of control system is that the killing process can simply be overcome through a
mutation which prohibits the killing function. One of the solutions for this is to
utilize two autonomous killing schemes, so that mutation of one scheme does not
overcome the other. This type of twofold scheme utilizes a plasmid with two lethal
genes encoding colE3 toxin as well as EcoRI restriction endonuclease. They are
killed through totally diverse processes, i.e. prohibition of protein synthesis and
degradation of DNA. In a true degradation experiment, these functions will lie side
by side with some genes like biodegradation genes, so that a deletion eliminating
colE3 as well as ecoRIR genes would also eliminate the gene to be regulated. The
12 Remediation of Heavy Metals Through Genetically Engineered Microorganism
351
3-methylbenzoate would not survive, either because of its degradation or because
of its escape to the environment, because of Gef expression. The escape rate from
killing in the lab was approximately 10
À8 cell
À1 generation
À1 . The testing of the
system was done in fields with bulk soil or rhizosphere soil and was very efficient.
There was no proof that it spread out of the experimental area (Torres et al. 2003a).
To evade lethal function loss because of mutation, double strains with extra function
depending upon the asd gene were made for diaminopimelic acid synthesis. This
gene is compulsory needed in cell wall synthesis, and strains without it need totally
external diaminopimelic acid, which is not present in the soil environments. A
P. putida strain with asd gene deletions has an alternative asd gene which is under
positive regulation by the XylS activator. This same XylS activator negatively
regulates gef expression too through transcription of the lacI gene as explained
earlier. Therefore, a strain devoid of 3-methylbenzoate would not survive in an
environment because of killing by Gef protein and also because of the absence of
diaminopimelic acid. The survival level of this strain due to the lack of
3-methylbenzoate was under the detection range (<10
À9 cell
À1 generation
À1
(Ronchel and Ramos 2001).
12.12.6 Suicide Mechanisms Preventing Horizontal Gene Transfer
The suicide scheme is created to allow reliable killing of recombinant strains from
being discharged into the wild. The same type of schemes could stop the transfer of
recombinant genes into other bacteria through conjugation, transduction or transformation. One diverse conditional lethal scheme was dependent on the colicin E3
(colE3) gene which kills several diverse bacteria through breaking of 16S ribosomal
RNA (Torres et al. 2003b). Under natural condition, this lethal function is opposed
through an immunity function (encoded by the immE3 gene) providing a poison/
antidote effect. The colE3 gene was kept inside a plasmid, and the immE3 gene was
put on the chromosome of E. coli or P. putida to test whether the horizontal transfer
of plasmid DNA to other bacteria could be stopped by using the killing function.
Therefore, horizontal transfer of the plasmid to other bacteria would cause cell death
in the recipient because the colE3 gene would be transferred without chromosomal
immE3 gene. This scheme has the capability to stop horizontal transfer into a broad
range of Gram-negative bacteria (Torres et al. 2003a). The main trouble with this
kind of control system is that the killing process can simply be overcome through a
mutation which prohibits the killing function. One of the solutions for this is to
utilize two autonomous killing schemes, so that mutation of one scheme does not
overcome the other. This type of twofold scheme utilizes a plasmid with two lethal
genes encoding colE3 toxin as well as EcoRI restriction endonuclease. They are
killed through totally diverse processes, i.e. prohibition of protein synthesis and
degradation of DNA. In a true degradation experiment, these functions will lie side
by side with some genes like biodegradation genes, so that a deletion eliminating
colE3 as well as ecoRIR genes would also eliminate the gene to be regulated. The
12 Remediation of Heavy Metals Through Genetically Engineered Microorganism
351