SURFkiller

The work with genetically modified organisms (GMOs) harbours the risk of their release to the nature. After release, serious consequences such as gene exchange with unmodified organism or ecosystem imbalance cannot be excluded. It is necessary to keep GMOs isolated from the environment or to make their survival in nature impossible. For this purpose, we decided to develop a security system for Bacillus subtilis. This system is based on one of the cells most important compartment- the ribosome. Ribosomes are essential as they are responsible for the protein synthesis and a loss of them leads to a halt of protein biosynthesis resulting in a rapid cell death.

To minimize the risk of failure of the SURFkiller as well as to ensure maximum safety we designed a system composed out of three interconnected modules which enable the survival of cells under laboratory conditions but not in the environment. All genes coding for components of our SURFkiller were deleted in the organism in order to guarantee a tight control of our system. Before deleting the genes the modules presented in figure 1 were inserted via homologous recombination into the genome. First of all module two was integrated into the amyE locus. Afterwards module two was placed into the lacA locus of B. subtilis, followed by the insertion of both, module 2 and 3 into the amyE locus.

Laboratory conditions and the environment are distinguished by the presence of IPTG (Isopropyl-β-D-thiogalactopyranosid), which is an artificial substrate for the LacI repressor. This compound is added to the medium in the laboratory but is absent in nature, leading to the activation of the SURFkiller system and cell death. This cell death is induced by the lack of the essential ribosomal L5 protein. This protein is essential for the assembly of the ribosomal subunits. In the absence of L5 the small and large subunits are synthesized but are not able to form functional ribosomes.

Fig. 1: Scheme of the SURFkiller system.
T4-Holin: Toxin -> lysis
T4-Antiholin: Antitoxin -> prevents lysis by Holin
YvyD: Hibernation factor like -> ribosomes not functional
L5: essential ribosomal protein

YvyD is affecting the ribosomes as well. In comparison to L5, YvyD is influencing already assembled ribosomes and inactivates them by dimerization. Those dimers are no longer capable of protein biosynthesis (Lauber et al., 2009). The expression of yvyD (module three) is under the control of a constitutive promoter, regulated by the TetR repressor which is produced by module one. Additional security is provided by synthesis of the antagonising toxin (T4-Holin; BBa_K112000) and antitoxin (T4-Antiholin). T4-Holin from the T4 phage is lysing the cells by introducing holes into the membrane. However T4-Antiholin, also from the T4 phage, is preventing this by plugging the holes (Wang et al., 2000; Young and Bläsi, 1995). If a mutation occurs in module two, the T4-Holin will immediately lyse the cells.

Figure 2: Effectiveness of Holin and Antiholin

The major regulator IPTG is inactivating the LacI repressor resulting in the constant activation of module one and two. In the case of an escape of the modified cells in nature, LacI is no longer inactivated and represses module one and two. This inactivation leads finally to cell death (Figure 3).

Figure 3: The SURFkiller in the lab and in nature

Lauber, M.A., Running, W.E., and Reilly, J.P. (2009) B. subtilis ribosomal proteins: structural homology and post-translational modifications. J Proteome Res 8: 4193–206 http://www.ncbi.nlm.nih.gov/pubmed/19653700. Accessed October 6, 2014.

Wang, I.N., Smith, D.L., and Young, R. (2000) Holins: the protein clocks of bacteriophage infections. Annu Rev Microbiol 54: 799–825 http://www.ncbi.nlm.nih.gov/pubmed/11018145. Accessed October 13, 2014.

Young, R., and Bläsi, U. (1995) Holins: form and function in bacteriophage lysis. FEMS Microbiol Rev 17: 191–205 http://www.ncbi.nlm.nih.gov/pubmed/7669346. Accessed October 14, 2014.