In order to socially implement our recombinant probiotics, we need to put safeguard in our engineered bacteria. So, we researched and discussed active containment using kill-switch. Then, we came up with the following proposal for that.
Metal ion inducible kill-switch
Active containment of genetically modified organisms using metal ion-inducible promoters is a system in which the promoter is repressed in the presence of certain metal ions, such as trivalent iron ions, but when the metal ions are removed, the gene encoding the toxin downstream of the promoter is expressed and the genetically modified organism commits suicide. The advantage of using iron ion-inducible promoters is that iron ions are safe for living organisms compared to cobalt and zinc ions.
However, ion-inducible promoters have only been successfully genetically modified in some algae, and there are still no successful examples in our lactic acid bacteria or other organisms. When these kill-switch carrying organisms are released into open ecosystems, they may survive in areas with locally high iron ion concentrations, and in the worst case, these may adversely affect non-genetically modified organisms. It is also difficult to continue feeding the trivalent iron ions liberated in the human body to an organism equipped with a kill switch.
Therefore, active containment of genetically modified organisms using metal ion-inducible promoters is so far only somewhat effective against genetically modified organisms that have accidentally escaped from a closed laboratory, and there are still problems to be solved before it can be used in the real world of open systems.
Hypoxia inducible kill-switch
One of our ideas is to insert genes that produce toxins and antitoxins against the engineered bacteria themselves. The gene encoding the antitoxin is placed downstream of a promoter that is always expressed, while the gene encoding the toxin is placed under the control of a promoter that is more strongly expressed in hypoxic conditions. This allows the kill switch to be triggered by hypoxia, preventing the spread of the recombinant bacteria.
The MazEF system, the one we are considering, uses MazF as a toxin and MazE as an antitoxin. The former acts as a site-specific endoribonuclease against almost all cellular mRNAs, while the latter neutralizes the action of MazF. The promoter that regulates MazF is BBa_K1720002, a promoter that works by hypoxia-inducible factor (HIF), and the promoter that regulates MazE is BBa_K747096, a CMV promoter.
The problem is, at what stage to create hypoxic conditions to activate the kill switch. There are not many hypoxic environments in nature, thus it is unlikely that the kill switch would be triggered spontaneously after the release of wastewater. In the past, wastewater was proposed to be collected and treated, but it is not practical to collect all of it, and if it leaks, it may lead to the spread of recombinants.
In our project, the engineered bacteria are acted upon in the aerobic environment of the oral cavity and then expelled through the anaerobic digestive tract. It is known that the partial pressure of oxygen in the rectal lumen is less than 1 mmHg (0.13% O2) . Also, the activity of HIF increases exponentially with decreasing oxygen concentration and shows a maximal response at 0.5% O2, which means that this kill switch can be fully activated in the rectum. Triggering it in the colon eliminates the risk of diffusion of the recombinant when it is expelled from the body.
Unnatural base pairs system
Unnatural base pairs system can be the strict containment because of its orthogonality. We came up with an idea of that. First, we introduce a gene into the lactic acid bacteria that produces a poison that kills itself. At this point, we do not use unnatural bases yet. The next gene to be introduced will be expressed by unnatural bases.
What kind of gene will be introduced? It is a gene that uses the supply or non-supply of the unnatural base as a switch for the life or death of the engineered lactic acid bacteria. In the first place, sequences containing unnatural bases will not replicate unless additional bases are supplied. We will apply this property.
Genes expressed by unnatural bases are aptamers that contain unnatural bases or proteins that contain unnatural amino acids corresponding to the unnatural bases. (We will refer to these aptamers and proteins as artificial aptamers and artificial proteins.) These artificial aptamers and proteins are made to neutralize the poison that was initially produced.
In other words, as long as the artificial bases are supplied to the engineered lactic acid bacteria, the internally produced toxins are neutralized by the artificial aptamers and proteins, and the engineered lactic acid bacteria can continue to live. However, if the supply of artificial bases is stopped, the artificial aptamers and proteins will not be produced and the engineered lactic acid bacteria will die from the poison produced by themselves. Therefore, the engineered lactic acid bacteria released into the environment cannot survive, and their spread will be prevented.
However, there are some problems that need to be considered. The first problem is that the sequence containing the unnatural base will be replaced by the natural base sequence when the supply of the unnatural base is stopped. In other words, if the aptamer containing the replaced natural base or the protein made by the replaced natural base has a non-toxic effect, it cannot be expected to prevent the spread of engineered lactic acid bacteria.
This can be solved by the properties of unnatural bases. First, I will explain about artificial aptamers. Many of the unnatural bases currently being developed are hydrophobic. In other words, by preparing a hydrophobic poison, aptamers containing hydrophobic unnatural bases can act on the poison, while aptamers containing only hydrophilic natural bases cannot. As for the artificial proteins, this can be solved by adding specific properties to the artificial proteins. Only artificial proteins that contain unnatural amino acids corresponding to unnatural bases can act on the poison, while proteins that contain only natural amino acids with natural bases cannot.
The second problem is how to supply unnatural bases to the engineered lactic acid bacteria living in the oral cavity. We suggest the use of gum. We propose the use of gum, which contains an unnatural base, to introduce the unnatural base to the engineered lactic acid bacteria in the mouth by making them chew the gum. If you want to keep the engineered lactobacilli alive in the mouth, chew the gum; if you want to kill them, stop chewing the gum. If the engineered lactobacilli escape from the mouth by swallowing or rinsing their mouths, they will not be supplied with the unnatural base and will die, thus preventing the spread of the engineered lactobacilli
Auxotrophy is the property of being unable to live without a certain substrate. By creating such a system, it is possible to prevent the engineered bacteria from living in nature by making it so that it can only live in a medium with a specific substrate added. As substrates to be dependent on, IPTG has been well studied, but we think that thymidine/D-alanine and polysorbate 80 are good ones to be used with lactic acid bacteria.
This is a well-studied technique, but it has the risk that mutations are likely to be a problem, and in the unlikely event that the required substrate exists in nature, it will survive.
However, there are also some problems that are common to all four of these.
1. Toxins to kill the engineered bacteria
In the mechanism of biocontainment, engineered bacteria will be killed by toxins which are produced by themselves. If the toxins have bad effects on the human body or natural environment, other problems will happen. This problem can be solved by using poisons that originally kill bacteria in the environment. For example, antimicrobial peptides originally found in the oral cavity can be used as poisons.
We must avoid mutations in the introduced gene impairing their ability to prevent the spread. The solution to this problem is to lay down multiple layers of bio containment technology. Even if one system is ceased, other functions can be used to stop the spread, thereby reducing the probability of mutation causing the diffusing. Also, by minimizing the number of genes, the slightest mutation can lead to lethality.
Each of these technologies has its advantages and disadvantages. We need to discuss what kind of safeguard mechanisms are suitable to realize our project. In order for synthetic biology to be useful in society in the future, we believe that safety and security must be guaranteed by strict biocontainment mechanisms.
 Lind Due V, Bonde J, Kann T, Perner A. Extremely low oxygen tension in the rectal lumen of human subjects. Acta Anaesthesiol Scand. 2003 Mar;47(3):372.
 B. H. Jiang et al. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. American Journal of Physiology-Cell Physiology 1996 271:4