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 nature?
Therefore, in almost all nations in the world the introduction of genetically modified organisms (GMOs) into the environment is strictly regulated[-1Robert] (for more information have a look at our talk with Dr. Ulrich Ehlers). 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 microorganisms. 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[-2Robert] 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 microorganism.
[EDIT] Kill switch variants and precursors [EDIT]
There are several different examples for the implementation of genetic kill switch systems from previous iGEM Teams. [1Simone, 2Simone, 3Simone]
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 can not 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. [4Simone] 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. [5Simone]
Figure 1: Schematic representation of the mazEF model [1Simone]
Various teams have used this system as a fundament and coupled it with their own ideas.iGEM Wageningen 2019, for example, has used the mazEF system together with the KaiABC system for circadian oscillations in gene expression. [1Simone]
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 iGEM 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.[2Simone]
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.[6Simone]
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.[7Simone] 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.[3Simone]
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.
[EDIT] Evolution of our kill switch design [EDIT]
We developed a concept for a kill switch design to hinder the wash-out of living organisms from the biofilm. Thereby our concept went through five phases. In this text we present our reasoning, show which decisions were based on which essential parameters and how the design of our kill switch has evolved over the course of the project.
Phase 1
Our initial idea for the basic design of a kill switch consisted of joining two strains in a communal biofilm. In each strain, an essential gene is knocked out in the genome and the lack of the gene is bridged by the expression of the respective gene from a plasmid. Essential gene expression is controlled by orthogonal promoter systems which are induced upon binding of small molecules produced by the respective communal partner strain. Consequently, the essential genes are only expressed if cells of the two strains are in close proximity to each other, which allows the transfer of inducing small molecules in the biofilm. Cells leaving the biofilm leave an environment with inducer concentrations enabling essential gene expression, which will eventually lead to cell death. (reference link)
Figure XY: Edit
Phase 2
After some research, we learned about the process of quorum sensing, which is already functional in various bacterial species (reference link). Quorum sensing is a fitting process for the function we want to create: First, the individuals of the biofilm are dependent on the community of cells. Strains that use quorum sensing produce autoinducers to regulate gene transcriptions. The amount of these autoinducers secreted by the cells is proportional to the cell density in the medium, because a higher cell density represents more cells that are capable of producing and secreting the inducer substance. If the concentration reaches a certain threshold concentration, the inducer is sensed by surrounding cells and this step initiates a phosphorylation cascade inside the respective cells. The cascade terminates with the activation of a regulator protein which finally alters the expression rate of the specific genes under control of quorum sensing
(siehe background).
Figure XY: Edit
Phase 3
Figure XY: Edit
Phase 4
Figure XY: Edit
Our final kill switch: design and function
When thinking about implementing “B-TOX” in wastewater treatment plants (WWTPs), we came to the realization that the majority of employees in WWTPs have never worked with genetically modified organisms (GMOs) before. GMOs are usually not to be found in WWTPs and since there are certain risks and a special safety protocol linked to the use of them, we made instructions on how to handle our final product “B-TOX”.
This manual contains a description of the risks of GMOs, a safety protocol and the practical application of “B-TOX” in the WWTP.
You can find it by clicking on the direction sign to the right.
Click me
Figure XY: Low cell concentration
Figure XY: irreversibility of the recombination
Which essential gene do we knock out?
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 . Since it encodes a protein that plays an important role in initiating DNA replication in prokaryotes
[1Jillian]
, its absence leads to cell death. However, at the end of the first phase of our kill switch design we noticed that the choice of this gene caused a problem: dnaA is located in an operon containing another gene
[1Jillian], [2Jillian]
. 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
[3Jillian]
, RpsB is essential for the functionality of the ribosomes and thus for translation. When a bacterium leaves the biofilm, RpsB will no longer be produced due to our killswitch. This leads to the bacterium being unable to keep up metabolic function, resulting in cell death. Another advantage is that rpsB is the only gene under control of the corresponding operon
[4Jillian]
, therefore being much easier to control.
Integration of our kill switch cassette
The implementation of additional, often heterologous, genes in organisms has always been a key step in Synthetic Biology. Several ways to insert new genes in organisms exist. Usually for prokaryotes, a plasmid with the genes of interest is introduced into the bacterium. With a corresponding origin of replication (ori), a fitting promoter region controlling the expression of the gene of interest, and an antibiotic resistance for selection, such a plasmid can often be very effective. Yet, several bacteria cannot robustly carry plasmids. In these cases, the genomic integration of the gene of interest is recommended.
Although B. subtilis can carry plasmids, its highly efficient homologous recombination machinery[1Robert]
allows the integration of our expression cassette in the genome. 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 (see Figure XY)[2Robert]. 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 advisors from the Kabisch Lab.
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.
Figure XY: Homologous Recombination
Critical aspects
There are many aspects to consider when designing and impelementing that have to be payed attention to in the design and implementation of a complex kill switch like ours.
First of all, the genetic code might not be stable over time.
Mutations, recombination and other cellular mechanisms may alter the genetic code.
All genetic circuits are susceptible to mutation.
A single point mutation could deactivate an entire circuit, rendering it inactive.
However, there have been improvements in genetic stability, especially in kill switches [1j].
Another problem depends on the Cre recombinase and its recombination sites.
If the recombination process loses its irreversibility, the whole kill switch could fail, as the constitutive promoter might get switched back in front of the essential gene.
That way, the essential gene is not under the control of the quorum sensing dependent promoter anymore, and the B. subtilis will not die when leaving the biofilm.
The possibility of this problem depends heavily on a mutation in the cre gene and the lox66 and lox71 recombination-site before first inversion [2j].
The safety of this kill switch also depends on whether the bacteria die after leaving the biofilm or only enter an inactive phase. We planned experiments to answer this question but because of the COVID-19 pandemic we were not able to execute them.
The fact that rpsB is an essential ribosomal protein leads to the presumption that the bacteria will die, as most likely its entire metabolic functions will collapse [3j].
Another potential downfall could be the comQXPA pathway itself.
Since it also conditionally required in sporulation processes [4j].
The question is whether the kill switch leads to infertile endospores. Due to the restrictions in 2020, we could not answer this question with experiments.
Future testing of the kill switch
Our concept of a kill switch is solely theoretical, because we were not able to work on the kill switch in the wetlab due to the COVID-19 pandemic. For the practical continuation of this project, it is essential that we can test the functionality of the kill switch. A critical parameter of the kill switch to characterize is for example the threshold of cell density at which the kill switch system is activated.
Most importantly, we have to ensure that our kill switch does what it is supposed to do: Killing our genetically modified Bacillus subtilis cells upon leaving the biofilm environment. We expect that the cells will die, if the essential ribosomal gene rpsB is not expressed, because many bacterial antibiotics inhibit the protein biosynthesis or ribosomal proteins. Thereby, some antibiotics are able to kill cells by blocking the activity of these important factors. [8Simone],[9Simone],[10Simone] Nevertheless, we cannot be fully sure whether knocking out the essential gene kills the cells or only inhibits their growth. Therefore, we have to test in the laboratory, if the cells are viable without the expression of the essential gene. This can be done via live/dead staining. In our planned assay, cells with damaged membrane, which are considered dead or dying, show a red staining, while cells with intact membrane are stained green upon incubation with SYTO 9/propidium iodide stain.
Further, we have to show experimentally, at which cell density the cells produce enough of the autoinducer ComX to be viable with the quorum sensing promotor PdeqQ solely controlling the expression of the essential rpsB gene. For this purpose, the cells are grown in separate test flasks up to a defined cell density. In the next step the expression of the recombinase is induced to initiate the irreversible inversion of the promotor region. Thereby the constitutive promoter in front of the gene is replaced by the ComX-dependent promoter. We hypothesize that if the cell density is not high enough, the induced switch of the promotor region would lead to the death of the cells. If it is the case, the cell density is high enough after the inducer has been set down for the growth phase, the cells should be able to live without a without an additional inducer. Cell viability and consequently the expression of the essential gene can be determined by live/dead staining, as already described above.
