Of great interest for an application of our system are the possible risks. The advantage of our system is that the cells grow in a closed system. A possible application would only be feasible in a closed cultivation system. As long as bacteria are protected by a mechanical barrier a cultivation is not restricted through the government. The use of GMOs is allowed for industrial applications since more than 20 years. This makes it easier to solve biosafety issues. Next to our main project we aim to develop a selection system without antibiotics. If the strain loses his plasmid it will die because of the missing alanine racemase which produces D-alanine. D-alanine is a key substance for the cell wall of bacteria which cannot be produced by our strain without the correct plasmid. In addition we did further research on a biosafety system which was developed by the iGEM Team Bielefeld-Germany 2013. It is dependent on rhamnose (Figure 1) because there are rhamnose promoters used in an alanine racemase deletion strain. This strain could be use as a chassis for heterologous gene expression. The alanine racemase produces D-alanine out of L-alanine which is a key substance for a bacterial cell wall. In this system the strain can not survive without the supplementation of rhamnose. In addition without rhamnose the RNase Ba (Barnase [Bacillus amyloliquefaciens]) will be expressed. This leads to lysis of the cell wall and degradation of the RNA (Figure 2) if the strain "escapes" the lab or loses his plasmid. Our strain is not dangerous in biosecurity terms because it has no pathogenicity. We only used bacterial strains known to be classified as security level 1 (S1). This is true for the bacterial chassis (E. coli) as well as for the heterologous expressed genes. These are also taken from organisms classified as S1. In our opinion our constructed strains have no relevant modifications which can be abused for bioterroristic purposes.
Further, our antibiotic free selection system reduces the possible threat of antibiotic resistant cassettes being transferred on wildtype strains during horizontal gene transfer. We worked on the implementation of the alanine racemase based antibiotic free selection system for everyday use in a wetlab.

Figure 1: With L-rhamnose the alanine racemase is expressed and the RNase Ba is inhibited.

Figure 2: Without L-rhamnose the expression of the alanine racemase is blocked which lead to cell lysis. In addition the RNase Ba cut the intracellular RNA.