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Since the beginnings of synthetic biology, there have always been a few questions to be raised when genetically modifying organisms: Do they pose a danger to humans, animals or the nature? Therefore, in almost all nations in the world, the introduction of genetically modified organisms (GMOs) into the environment is strictly regulated (link zu rechtliches, HP). There are good reasons for this: Genetically modified, non-native organisms can have completely unforeseen effects when entering a novel ecosystem. As an example, an inserted antibiotic resistance might be passed on to other bacteria, leading to multiple resistances. This raises the important question: How do we prevent these organisms or their recombinant genetic material from entering the environment?
Most importantly when working with GMOs, strict attention must be paid to the so-called biocontainment. In general, GMOs are not supposed to leave the designated facility. In the laboratory, disinfection or autoclaving of the equipment are the most common methods to efficiently neutralize all remaining organisms. Working inside of special rooms like cleanrooms or at a sterile bench can not only improve the quality and purity of a product, but can further reduce the risk of a GMO escaping the facility.
In contrast to the physical and chemical methods previously mentioned, there is also the option for a biological containment method, called kill switch. A kill switch is a genetic circuit used for selective elimination of a GMO. The GMO is supposed to stay in its defined area. When it leaves the defined area, a genetic switch is triggered, leading to the death of the organism.

The implementation of foreign genes in organisms has always been a key step in Synthetic Biology. There are several ways to insert new genes in organisms. Usually for procaryotes, a plasmid with the genes of interest is introduced into the bacterium. With a corresponding origin of replication (ori), a fitting promoter region, and an antibiotic resistance for selection, such a plasmid can be very effective. However, there are also other ways to implement our cassette. B. subtilis has a highly efficient homologous recombination machinery ^[1Robert] . The cell normally uses this mechanism to repair DNA double-strand breaks, by using a homologous DNA fragment as a blueprint. This machinery also works without the generation of double-strand breaks. Therefore, genes of interest can be integrated directly into the genome of B. subtilis. Only a plasmid with double-stranded DNA, containing our genetic cassette, an antibiotic resistance for selection and homologous sequences to the genome, are required for insertion ^[2Robert] (see Fig. 1). Theoretically a homology of ~70 base pairs is sufficient, however, we decided to use around 500 base pairs of homologies for both flanks after talking to our supervisor. We decided to use this technique to integrate our kill switch cassette into the genome of B. subtilis. This additionally prevents our cassette from being lost during replication, which is a major problem when using plasmids, as a constant selection pressure has to be maintained.

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There are several different examples for the implementation of genetic kill switch systems from previous iGEM Teams. [1, 2, 3]
In previous iGEM projects one popular approach was the utilization of toxin-antitoxin systems:
This system, as the name suggests, consists of two components: the toxin and the corresponding antitoxin. The two genes that encode these components may be present on a plasmid. If the cells are not under selection pressure, the plasmid will be lost in the next generation and they will die, because the gene that encodes the antitoxin is not present. Thus the unstable antitoxin cannot be produced an is degraded and the stable toxic protein kills the new cell.
A well-researched tox-antitoxin system is the mazEF system in E. Coli. But there are also mazEF -like modules in the genome of many other bacteria including pathogens. [4] Its natural function is to prevent the spread of a bacterial phage infection. MazF is a sequence-specific RNA endoribonuclease and thus a stable toxin that initiates a programmed cell death pathway. MazE is the unstable antitoxin and binds MazF. Figure 1 shows a schematic representation of the system. [5] Various teams have used this system as a fundament and coupled it with their own ideas. Wageningen 2019, for example, has used the mazEF system together with the KaiABC system for circadian oscillations in gene expression. [1]
One common problem with this system is the constant expression of the toxin gene due to promoter leakiness leading to unwanted cell death.
A second example for the use of the mazEF system is provided by Munich 2012. They used the mazEF system in combination with a sigma factor G to kill sporulating B. subtilis. In this case, the main issue of this project was that the promoter was too strong for the toxin and therefore there was an overproduction of MazE. [2]
Another popular approach is autotrophy: One way to overcome the difficulties of the toxin-antitoxin system is to knock out an essential gene. Essential genes are genes that encode for components that are vital for the life of the cell. If such a gene is destroyed or knocked out, the cell can only survive if it can obtain the missing component from other sources. If the bacteria escape into the environment, they can not survive due to the lack of essential molecules. [6]
iGEM NCKU Tainan 2015 used this method to switch off the dapA gene. The gene product of dapA is 4-hydroxy-tetrahydrodipicolinate synthase. This enzyme is a part of the diaminopimelate pathway and therefore plays an important role in cell wall synthesis. [7] If this gene is knocked out, no synthesis of diaminopimelate (DAP) takes place and the cells can only grow via an external supply of DAP. If the cell leaves the controlled environment, it will not survive. [3]
However, this approach was not possible for our project, as these components cannot be easily added in the wastewater treatment plant. The reason for this is that the components would be flushed away, before they could enter the cells. Therefore, large amounts of essential molecules would have to be used, which is not cost-effective and not practically feasible. Furthermore, these molecules would then float in the water, so there would be no guarantee that the microorganisms would actually die after leaving the biofilm.
Therefore, we thought of something else: our approach is based on the idea of an inducible gene switch to control an essential gene. The essential gene is genetically modified in a way that it can be controlled by an inducible promoter. In the area of application of the biofilm the molecules necessary for the transcription of the essential gene are present, the protein is synthesized and the cell is viable. If the cell leaves this application area, the necessary molecules are missing and the cell cannot synthesize the essential components and the bacterium cannot survive.

Since our kill switch is based on linking the availability of an essential gene (product) to the presence of a biofilm, this essential gene first had to be knocked out. In our first approach we chose the gene dnaA for this. Since it encodes for a protein that plays an important role in initiating DNA replication in prokaryotes ^[1Jillian] , its absence would lead to cell death. However, after some time we realized that the choice of this gene caused a problem: dnaA is located in an operon that contains other genes ^[1Jillian] . In the case of a knockout by destroying the promoter, the entire operon would no longer be transcribed. This could have led to unpredictable complications.

In addition, dnaA is only essential for cell division, so it is possible that the microorganisms do not die but go into dormancy instead. For this reason, we searched for an easier accessible gene - and found rpsB. Its gene product is the ribosomal protein S2 in the 30S subunit of prokaryotic ribosomes. As one of the biggest ribosomal proteins in the small subunit ^[2Jillian] , RpsB is essential for the functionality of the ribosomes and thus for translation. When a bacterium leaves the biofilm, due to our killswitch RpsB will no longer be produced. This leads to the bacterium being unable to keep up metabolic function, resulting into a fast cell death. Another advantage is that rpsB is the only gene under control of the corresponding operon, therefore being much easier to control.