Currently, numerous scientific groups are actively engaged in genetic engineering, with applications ranging from using genetically engineered bacteria inside the human body for disease diagnosis and treatment to deploying them in natural environments for pollution remediation. However, the introduction of genetically modified organisms (GMOs) into their intended settings necessitates the development of stringent containment measures to prevent potential environmental risks and uncontrolled proliferation.

To address this challenge, scientists have been focusing on the creation of 'kill switches'—genetic circuits designed to ensure biocontainment by preventing the unintended growth of engineered microorganisms through the expression of toxin genes. This year, our project, Sarcotreat, involves the development of an engineered biotherapeutic product designed to combat sarcopenia. Given that Sarcotreat will be ingested by humans and eventually released into the environment via human fecal matter, there is a genuine concern about the potential environmental impact. Engineered microorganisms, if left unchecked, could pose threats to ecosystems, potentially outcompeting native organisms or causing significant changes in the gene pool. In light of these concerns, the use of a kill switch is not justifiable but essential to ensure responsible and safe deployment of our engineered biotherapeutic product.

General design

Hydrogen sulfide, a gas present in the intestine, inspired our use of it as an indicator to regulate our kill switch. In this approach, if our engineered organism escapes containment, it can sense the low hydrogen sulfide concentration in the external environment and subsequently undergo cell death.

SqrR, a member of the arsenic repressor (ArsR) family, plays a crucial role in responding to hydrogen sulfide and reactive sulfur species (RSS). SqrR binds to the promoter in the absence of hydrogen sulfide, inhibiting transcription. Conversely, it permits transcription in the presence of hydrogen sulfide.

Leveraging our understanding of SqrR, we initially designed a two-gear kill switch, as outlined below

However, we encountered two significant challenges in our design process:

As we are developing Life Bio-Therapeutic Products (LBPs), our intention is to encapsulate our modified organisms in medicinal capsules. A two-tiered kill switch system posed a problem because it would lead to the immediate demise of our organisms inside the capsules due to the absence of hydrogen sulfide.

We also had concerns about the strength of the constitutive promoter responsible for driving toxin production, which could potentially overpower the antitoxin and result in the demise of our organisms.

To address these issues, we proposed a three-gear kill switch solution. We incorporated the DRE recombinase to control our system, resolving the first problem. Additionally, we employed an oxygen-sensitive promoter to regulate toxin expression, tackling the second challenge. In an environment with high oxygen levels, such as outside the body, the toxin promoter becomes more active, ensuring that our engineered organisms are effectively eliminated. In contrast, within the anaerobic environment of the gut, the promoter's activity is reduced, preventing an excess of toxin production and ensuring a balance with the antitoxin.

We have formulated a general 'formula' for the kill switch of our two organisms, which is detailed below..

Kill switch’s mechanism

In Figure 3, we depict the initial exposure to normal conditions. In this scenario, when hydrogen sulfide levels are low, the translated SqrR protein (red) effectively represses the sqr promoter, resulting in the prevention of transcription for recombinases DRE or CRE ( gray) and the antitoxin (pink). Furthermore, the presence of terminator sequences labeled by rox or Lox sequences puts a halt to the transcription of the toxin. Consequently, under these conditions, the absence of both toxin and antitoxin prevents any cell death.

In the second condition, as illustrated in Figure 4 and 5, when the organism is exposed to the gut environment characterized by the presence of hydrogen sulfide (depicted as one yellow dot with two blue dots), the suppressive effect of the SqrR protein (red) decreases. This reduction in suppression leads to the transcription of recombinase DRE or CRE (gray) and the antitoxin. The recombinase, in turn, targets the terminator sequence labeled by rox/Lox and cleaves it, enabling the transcription of the toxin(blue). However, it's important to note that the transcribed antitoxin (pink) counteracts the toxin, neutralizing its toxic activity. In conclusion, under these conditions, the presence of both toxin and antitoxin allows the cell to remain viable.

In the third condition, depicted in Figure 6, we illustrate the organism's return to the normal environment, indicating its escape from the gut or excretion of feces. Under these circumstances, the SqrR suppressor (depicted in red) once again binds to the promoter, effectively inhibiting the transcription of the antitoxin. However, crucially, the terminator that normally halts toxin transcription is excised. This excision allows for the transcription of the toxin. In this scenario, the bacteria lose the protective antitoxin, leading to cell death as a consequence.

Bacillus subtilis

We tailored our kill switch for Bacillus subtilis, selecting MazF as the toxin and MazE as the antitoxin, with DRE serving as our recombinase. Additionally, we carefully chose a promoter and terminator that are compatible with Bacillus subtilis, ensuring the effectiveness of our engineered system. This kill switch is shown at BBa_K4743030.

Kluyveromyces marxianus

While the core concept of the kill switch remains consistent for Bacillus subtilis, we have undertaken specific adaptations tailored to Kluyveromyces marxianus. Our foremost adjustment involves the replacement of the recombinase with CRE, and we have meticulously tailored the promoters and terminators to align with the distinct genetic characteristics of Kluyveromyces marxianus, which differ from those of Bacillus subtilis. Furthermore, we've segmented the composite kill switch into four distinct sections, each under the control of its unique promoter. This modification is imperative because Kluyveromyces marxianus operates under a monocistronic pattern, contrasting with the polycistronic pattern observed in Bacillus subtilis.

To address the issue of promoter compatibility-That is, prokaryotes promoter can’t be performed by eukaryotes. We have integrated two promoters into one, combining Promoter ADH1 with Promoter sqr. The ADH1 promoter serves as the driver for RNA polymerase, while the sqr promoter takes on a regulatory role by facilitating the binding of the sqrR repressor. It's worth noting that this composite promoter is currently a hypothesis and requires further experimental validation to ensure its functionality and compatibility within the Kluyveromyces marxianus system.


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