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Team:Bielefeld-CeBiTec/Project/Biosafety
From 2014.igem.org
Biosafety - Antibiotic-free Selection
We work on two different biosafety systems.
First of all we aim to implement an selection system without antibiotics. The target of this system is to perform two deletions in the E. coli KRX genome for two genes encoding the alanine racemase. The alanine racemase catalyses the reversible reaction from L-alanin into D-alanin. D-alanin is an important component of the bacterial cell wall. Every plasmid which is used for the transformation should carry the alanine racemase to complement the deletion. Without the uptake of the plasmid the bacterial cells will die because of their lack of cell wall production.The advantage of this system is visible during the scientific research. It can be used for all cloning and selection purposes in labs all around the world. We also work on a second biosafety system which is based on two steps.
For second system the supplementation with L-rhamnose is essential. In the presence of L-rhamnose a rhamnose dependent promotor is activated which leads to the expression of araC and the alanine racemase. The protein of araC inhibits the expression of the PBAD promotor. In the absence of L-rhamnose two kill switches are activated. If the rhamnose dependent promotor is not activated D-alanine could not be produced which lead to the lysis of the cell wall. In addition araC is not expressed anymore. This leads to the expression of the RNase Ba (Barnase) which leads to the lysis of intracellular RNA.
Listeria monocytes (Thompson et al., 1998), Corynebacterium glutamicum (Tauch et al., 2002) or Bacillus subtilis (Ferrari et al., 1985)
First of all we aim to implement an selection system without antibiotics. The target of this system is to perform two deletions in the E. coli KRX genome for two genes encoding the alanine racemase. The alanine racemase catalyses the reversible reaction from L-alanin into D-alanin. D-alanin is an important component of the bacterial cell wall. Every plasmid which is used for the transformation should carry the alanine racemase to complement the deletion. Without the uptake of the plasmid the bacterial cells will die because of their lack of cell wall production.The advantage of this system is visible during the scientific research. It can be used for all cloning and selection purposes in labs all around the world. We also work on a second biosafety system which is based on two steps.
For second system the supplementation with L-rhamnose is essential. In the presence of L-rhamnose a rhamnose dependent promotor is activated which leads to the expression of araC and the alanine racemase. The protein of araC inhibits the expression of the PBAD promotor. In the absence of L-rhamnose two kill switches are activated. If the rhamnose dependent promotor is not activated D-alanine could not be produced which lead to the lysis of the cell wall. In addition araC is not expressed anymore. This leads to the expression of the RNase Ba (Barnase) which leads to the lysis of intracellular RNA.
Listeria monocytes (Thompson et al., 1998), Corynebacterium glutamicum (Tauch et al., 2002) or Bacillus subtilis (Ferrari et al., 1985)
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.
