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Team:Bielefeld-CeBiTec/Project/Biosafety
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Biosafety
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 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 factor for 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. No antibiotics have to be used for fermentations for example. Therefor we 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 lead 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.
By using a closed system and working with safety strains considering specifications our project fulfills regularities for an industrial application. The question of biosecurity terms is manifold. The use of BioBricks enables an easy generation of modified organisms by combining different parts. The parts we are using during our project are taken from safety level 1 organisms. At this moment there are no hints for a possibility to abuse parts of our project for terroristic aspects (Dr. Arnold Sauter).
Here you will find the results of our biosafety.
First of all we aim to implement an selection system without antibiotics. The target of this system is to perform two deletions in the 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 factor for 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. No antibiotics have to be used for fermentations for example. Therefor we 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 lead 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.
By using a closed system and working with safety strains considering specifications our project fulfills regularities for an industrial application. The question of biosecurity terms is manifold. The use of BioBricks enables an easy generation of modified organisms by combining different parts. The parts we are using during our project are taken from safety level 1 organisms. At this moment there are no hints for a possibility to abuse parts of our project for terroristic aspects (Dr. Arnold Sauter).
Here you will find the results of our biosafety.
Instruction manual GenTSV (S1) 
Instruction manual GefStoffV
