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Team:Bielefeld-CeBiTec/Project/Biosafety
From 2014.igem.org
Biosafety - Antibiotic-free Selection
An import aspect of synthetic biology is the prevention of interactions between genetically modified bacteria and natural wild type strains. This prevents the uncontrolled distribution of synthetic sequences in the enviroment. Even if synthetic DNA would be released from a laboratory it would be very unlikely that it is integrated in a bacterial genome. Most organisms protect themselves against foreign nucleic acids for example by expressing special RNases and DNases (Bertani and Weigle, 1953; Tock and Dryden, 2005) and apart because the genetically modified bacteria are adapted to the excellent conditions of the laboratory, the natural bacteria will usually outlast these modified strains in nature due to their better adaptation to their environment. But there is no guarantee that there is really no interaction and that their release does not affect the equilibrium of the environment .
So the question how a novel designed sequence or even a heterologous used sequence would affect the environment, can be divided into three cases. In the first scenario it is an artificial sequence of DNA or even a protein, that does not occur in nature and does not interact with the normal way of live, because of an other DNA structure like XNA as example. For this case it might be save to exclude interaction with the environment.
The second case deals with sequences that are useless for the survival of an organisms in his natural enviroment, like the RuBisCO we brought into E. coli, which is only useful within high concentration of carbon dioxide and the supply with ribulose 1,5 bisphosphate or the Fumerate reductase, which also might be only useful under special conditions of an increasing electron supply. So assumed that an intact sequence of one of this genes would get into a natural cell, it might be lost even faster because of its higher burden to the cell.
The third case are the type of genes that probably have an greater impact to the environment, like antibiotics and other bacterial or artificial toxins that entail an advantage in selection for the bacteria. Because of this, the uptake of a corresponding sequence will have an impact of the environment. Toxin encoding sequences are for example used in kill switches, which should prevent genetically modified bacteria from escaping the containment. If these systems fail there is the possibility of the distribution of such sequences in the enviroment. Also the probability is very low the consequences would be severely. A insensitive bacterium poses a great risk to its enviroment if it starts producing a toxin. The consequences could be even more striking then that of a antibiotic resistance in the enviroment. It is well known that antibiotic resistances are spreading out due to varies reasons (Heuer et al., 2011; Lupo et al., 2012). This increases the risk of the formation of multi resistances, which causes problems in medical facilities, where many weak people are concentrated. Antibiotic resistances occur in nature in many species. The distribution of such resistances is a novel research field. Soil bacteria seem to be an reservoir of antibiotic resistance genes (Riesenfeld et al., 2004; Forsberg et al., 2012). All antibiotic resistances which are used in the laboratory were isolated from somewhere in the enviroment. Nevertheless it is crucial to keep these genes from spreading out.
To adress this problem we implemented an antiobiotic-free selection system by the complementation of a D-alanine auxotrophic E. coli strain to reduce the need for antibiotic resistances as selection markers and enable an antibiortic-free plasmid selection.
And on top this sysem might be not only limited to E. coli since a D-alanine auxotrophy could be demonstrated also for other bacteria like Listeria monocytes (Thompson et al., 1998), Corynebacterium glutamicum (Tauch et al., 2002) or Bacillus subtilis (Ferrari et al., 1985) as example, so that it might be feasible with any bacteria where the cross-linkage of the peptidoglycane layer is realized with D-alanine...
The second case deals with sequences that are useless for the survival of an organisms in his natural enviroment, like the RuBisCO we brought into E. coli, which is only useful within high concentration of carbon dioxide and the supply with ribulose 1,5 bisphosphate or the Fumerate reductase, which also might be only useful under special conditions of an increasing electron supply. So assumed that an intact sequence of one of this genes would get into a natural cell, it might be lost even faster because of its higher burden to the cell.
The third case are the type of genes that probably have an greater impact to the environment, like antibiotics and other bacterial or artificial toxins that entail an advantage in selection for the bacteria. Because of this, the uptake of a corresponding sequence will have an impact of the environment. Toxin encoding sequences are for example used in kill switches, which should prevent genetically modified bacteria from escaping the containment. If these systems fail there is the possibility of the distribution of such sequences in the enviroment. Also the probability is very low the consequences would be severely. A insensitive bacterium poses a great risk to its enviroment if it starts producing a toxin. The consequences could be even more striking then that of a antibiotic resistance in the enviroment. It is well known that antibiotic resistances are spreading out due to varies reasons (Heuer et al., 2011; Lupo et al., 2012). This increases the risk of the formation of multi resistances, which causes problems in medical facilities, where many weak people are concentrated. Antibiotic resistances occur in nature in many species. The distribution of such resistances is a novel research field. Soil bacteria seem to be an reservoir of antibiotic resistance genes (Riesenfeld et al., 2004; Forsberg et al., 2012). All antibiotic resistances which are used in the laboratory were isolated from somewhere in the enviroment. Nevertheless it is crucial to keep these genes from spreading out.
To adress this problem we implemented an antiobiotic-free selection system by the complementation of a D-alanine auxotrophic E. coli strain to reduce the need for antibiotic resistances as selection markers and enable an antibiortic-free plasmid selection.
And on top this sysem might be not only limited to E. coli since a D-alanine auxotrophy could be demonstrated also for other bacteria like Listeria monocytes (Thompson et al., 1998), Corynebacterium glutamicum (Tauch et al., 2002) or Bacillus subtilis (Ferrari et al., 1985) as example, so that it might be feasible with any bacteria where the cross-linkage of the peptidoglycane layer is realized with D-alanine...
References
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E. Ferrari, D. Henner und M. Yang (1985) Isolation of an alanine racemase gene from Bacillus subtilis and its use for plasmid maintenance in B.subtilis. Nature Biotechnology, vol. 3, pp. 1003 - 1007.
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A. Tauch, S. Götker, A. Pühler, J. Kalinowski, G. Thierbach (2002) The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains. Journal of Biotechnology, vol. 99, pp. 79 - 91.
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R. Thompson, H. Bouwer, D. Portnoy, F. Frankel (1998) Pathogenicity and and immunogenicity of a Listeria monocytogenes strain that requires D-alanine for growth. Infection and Immunity, vol. 66, pp. 3552 - 3561.
