non-conjugative plasmids like RSF1010, generally employed in genetic modification with Pseudomonas, keeps the oriT region which allows their perfect transfer
under the presence of self-transmissible plasmids like RP4. The plasmid can still be
transferred through the making of cointegrate which utilizes homology of common
DNA sequences, even if the oriT region is not present. Various efforts to make
vectors with the focus on the biodegradation field-release targets on defective
transposons which can be incorporated only once into the chromosome and cannot
be used again because of the absence of the transposase gene. Genes incorporated in
the minitransposons exhibited a very low degree of horizontal transfer (<10
À9 cell
À1
generation
À1 (Torres et al. 2003b).
12.12.4 Removal of Antibiotic Resistance Genes
It is an exceptional event; therefore, transposition vectors need very powerful
selective marker generally antibiotic resistance. But, on biosafety grounds for
releasing into the environment, an antibiotic marker is not required. Several
transposons with resistance for zinc, mercury or tellurite have also been planned
(Davison 2002a, b), but they can permit selective advantage in certain conditions. As
the selective markers are utilized only to discover the presence of transposition, the
easiest solution is to cut it afterwards through a site-specific recombination event
(Davison 2002b; Schweizer 2003). Such contemporary procedures permit the cutting of DNA sequences present in between two site-specific recombination sites like
loxP or FRT after transient expression of the recombinase (cre or FLP). In one
instance, an antibiotic resistance marker was removed through the site-specific ResA
resolvase; therefore, recombinant bacteria possess only the lux genes essential to
monitor the environment. Besides improving biosafety concerns, this technique
permits reuse of selectable markers for sequential strain productions.
12.12.5 Suicide Mechanisms Preventing Escape of Recombinant
Bacteria
Even implementation of defective transposons has the possibility of horizontal
transfer through recombination of conjugative plasmid or through bacterial conjugation. Therefore, several vectors are created which possess extra safety measures
through the use of a large number of suicide processes (Davison 2002a). In the most
simple way, suicide may be inhibited through an environmental signal such as the
pollutant which has to be degraded, permitting cell survival. The absence of this
signal causes induction of the suicide gene, which results in cell death. In one of the
best-studied instances, the lethal gef gene carrying a mini-Tn5 transposon was
regulated by the LacI repressor. Therefore, cell survival is dependent on the continuous existence of this negative control factor. On the contrary, transcription of the
lacI gene itself is dependent upon the positive-activator XylS of the meta-cleavage
pathway which is active under the presence of 3-methylbenzoate. Therefore, the
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N. Srivastava
under the presence of self-transmissible plasmids like RP4. The plasmid can still be
transferred through the making of cointegrate which utilizes homology of common
DNA sequences, even if the oriT region is not present. Various efforts to make
vectors with the focus on the biodegradation field-release targets on defective
transposons which can be incorporated only once into the chromosome and cannot
be used again because of the absence of the transposase gene. Genes incorporated in
the minitransposons exhibited a very low degree of horizontal transfer (<10
À9 cell
À1
generation
À1 (Torres et al. 2003b).
12.12.4 Removal of Antibiotic Resistance Genes
It is an exceptional event; therefore, transposition vectors need very powerful
selective marker generally antibiotic resistance. But, on biosafety grounds for
releasing into the environment, an antibiotic marker is not required. Several
transposons with resistance for zinc, mercury or tellurite have also been planned
(Davison 2002a, b), but they can permit selective advantage in certain conditions. As
the selective markers are utilized only to discover the presence of transposition, the
easiest solution is to cut it afterwards through a site-specific recombination event
(Davison 2002b; Schweizer 2003). Such contemporary procedures permit the cutting of DNA sequences present in between two site-specific recombination sites like
loxP or FRT after transient expression of the recombinase (cre or FLP). In one
instance, an antibiotic resistance marker was removed through the site-specific ResA
resolvase; therefore, recombinant bacteria possess only the lux genes essential to
monitor the environment. Besides improving biosafety concerns, this technique
permits reuse of selectable markers for sequential strain productions.
12.12.5 Suicide Mechanisms Preventing Escape of Recombinant
Bacteria
Even implementation of defective transposons has the possibility of horizontal
transfer through recombination of conjugative plasmid or through bacterial conjugation. Therefore, several vectors are created which possess extra safety measures
through the use of a large number of suicide processes (Davison 2002a). In the most
simple way, suicide may be inhibited through an environmental signal such as the
pollutant which has to be degraded, permitting cell survival. The absence of this
signal causes induction of the suicide gene, which results in cell death. In one of the
best-studied instances, the lethal gef gene carrying a mini-Tn5 transposon was
regulated by the LacI repressor. Therefore, cell survival is dependent on the continuous existence of this negative control factor. On the contrary, transcription of the
lacI gene itself is dependent upon the positive-activator XylS of the meta-cleavage
pathway which is active under the presence of 3-methylbenzoate. Therefore, the
350
N. Srivastava