Kill Switch
In order to ensure our engineered probiotic will not jeopardize the environment or users, this year, we integrated two kinds of Kill Switches into our project. The major kind of Kill Switch is cold triggered toxin/antitoxin Kill Switch to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine.
Why our project need kill switches ?
Kill Switch is a gene device to eliminate bacteria in a short period of time when engineered bacteria escape from the set conditions. Kill Switches commonly are applied on account of three reasons, hindering gene vertical transfer, hindering gene horizontal transfer and preventing the survival of bacteria outside certain environments [1].
The ultimate goal of our project is to produce a commercial calcium supplementary probiotic colonized in the intestines. An eco-friendly commercial probiotic should avoid the occurrence of modified gene vertical transfer and spread widely in the natural environment when excreted from the human intestine. And gene vertical transfer is directly associated with the survival of bacteria. Therefore, we needed to construct a Kill Switch to deprive of the survivability of engineered E. coli outside human bodies.
Simultaneously, we noticed that consumers may want to cease probiotics functioning because of accidental overdose, incidental gastrointestinal distress or any other reasons. We decided to qualify our probiotic for rapid suicide by BBa_K3036005.
Also, concerning hindering gene horizontal transfer, we provided a potential solution other than introducing one more Kill Switch. (Click here to see our solution on the Future Expectation of Safety page.)
Learn from Previous experience
Previous experience is a very important source to learn from. So, before we formally started to design our own Kill Switch, we traversed most suicide circuits in intestinal probiotics by iGEM teams and summarized mostly used methods. The Kill Switch database made by team 2016 Marburg has incredibly reduced our workload in searching Kill Switches used from 2007 to 2015. (Click here to see the database.)
After summarizing, we discover that the most commonly used kill switch circuit can be divided into two core components, the killer system and the sponsor system. According to the concept of biobrick in synthetic biology, we catalog parts of the killer system and the sponsor system separately. For parts of resemble mechanism, only the latest and the most efficient ones are listed. In this way, we can easily figure out the fittest part in the two groups to compose our kill switch, and hopefully offer a reference table for the future iGEM team working on intestinal probiotics to design kill switch and choose parts more conveniently.
Table 1. summary of the mostly used killer system in intestinal probiotics project. More “+” represents higher efficiency.
| Killer System | Advantages | Disadvantages | Parts | Efficiency | Comments |
|---|---|---|---|---|---|
| Toxin | easy, direct | The leakage of toxin may cause engineered E. coli lysing in the body and interrupting native flora. | MazF cas3 Phi 174 Gene E protein | +++ | Expression of toxins alone have been shown to help develop bacterial persistence [2]. |
| Toxin/antitoxin system | stable, flexible | / | MazF/MazE RelE/RelB | ++++ | TA systems can stabilize plasmids and facilitate rapid adaptation of gut without negative effect on fitness of the human host. [1] |
| Vital gene knock out | efficient | non-universal, time-consuming for testification. | can gene knock out Deactivating Exendin-4 expression system by cas3 | ++++ | Note 1 |
Note 1: The method of can gene knock out by last year’s grand prize winner is ingenious and effective, unfortunately, it can be applied to our project. It is reported that carbonic anhydrase, expression product of can gene, influence the release of PTH, leading to complex effects on bone remodeling. Carbonic anhydrase deficiency is manifested by osteopetrosis in human.[2] Although no concrete evidence has been found to show the relevance between carbonic anhydrase deficiency in intestinal microbiota and osteopetrosis, we decided to give up this method to avoid potential problems.
Table 2. summary of the mostly used response system in intestinal probiotics project. More “+” represents higher efficiency and more “-” represents stronger killing power.
| Physical Parameters | Response System | Sensitivity | Killing power | Parts | Comments |
|---|---|---|---|---|---|
| Temperature (cold shock) | Cold-acting promoter | ++ | --- | PcspA | Working efficiently only below the room temperature (20℃). |
| cI repressor system | +++ | --- | cI repressor lambda cI regulated promoter TCI38 TCI | Kill bacteria efficiently only when reaching 40℃, so it is not appropriate for our project that expect E. coli to survive at that temperature. Moreover, it requires to express two types of protein which imposes more burden to the bacteria. | |
| RNA thermometer | +++++ | ---- | FourU NoChill-06 | Appropriate reaction temperature range, high sensitivity and high efficiency. | Concentration of essential nutrients in the intestine | glucose-sensitive promoter | ++ | --- | rpoH P5 PT-αCRP | Glucose starvation promoter (BBa_K3142012) initiate transcription efficiently with concentration of glucose lower than 0.05%. The concentration of glucose in small intestine will reduce to 0.05% two hours after dinner and limit the survive of E. coli. |
| Fatty acid responsive repression system | + | -- | Fatty acid responsive repression system | Metabolites as responding factors, are greatly affected by food intake. | Other different conditions | N-Acetyl-Glucosamin-6 phosphate regulated repression system | + | -- | N-Acetyl-Glucosamin-6 phosphate regulated repression system | It is not very efficient. |
| CO2 concentration | ++ | ---- | can gene knock out | Note 1 | |
| light condition | + | - | killer red | It is of very low efficiency and the demand of light illumination greatly limited its application contexts. |
Design Our own Kill Switches
After a throughout analysis of the above two tables, we finally selected the toxin/ antitoxin system from killer systems and RNA thermometer from the response systems.
The toxin/antitoxin system is a mature and stable system that is extensively applied both inside and outside the iGEM competition. By regulating the strength of RBS, we could achieve a state that toxin is totally neutralized and guarantee the security on the host. Here we first chose RelE/RelB system of type II TA systems but replace it with MazF/MazE system later. (Click here to see our consideration in engineering.)
RNA thermometers are apparent outstandingly because of its appropriate reaction temperature range, high sensitivity and high efficiency. Moreover, they do not rely on extra proteins or are affected by food intake. Here we selected NoChill- 06 which presents to be the most sensitive and efficient one.
So, our Kill Switch consists of a toxin-antitoxin system and an RNA thermometer NoChill-06 to regulate it to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine.
Figure 1. cold triggered MazF/MazE Kill Switch under body temperature(37℃) and outside the body(30℃)
The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer No-Chill 06. Under the body temperature (37℃)，No-Chill 06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio. When the bacteria encounter a cold shock(30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP and releases MazF. The toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, therefore resulting in cell growth arrest and finally cell death [5]. The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer No-Chill 06. Under the body temperature (37℃)，No-Chill 06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio. When the bacteria encounter a cold shock(30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP and releases MazF. The toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, therefore resulting in cell growth arrest and finally cell death [5].
In the former version of RelE/RelB system, the toxin RelE is a global translational inhibitor that cleaves mRNAs under translating at the ribosomal A site and the antitoxin RelB is degraded rapidly by an ATP-dependent serine protease Lon [6].
Demonstrate the Kill Switches
Model(补图)
According to the special situation this year, we have limited time to work in the wet lab. As the Kill Switch model is relatively independent of our main project, we convert to establish the demonstration of the Kill Switch on pure dry lab work. We find out that our Kill Switch design has a very similar basic idea with BNU-China, so we decide to establish collaboration with them. We adopt their raw wet lab experimental data of RelE/RelB system to construct our model.
We improved the logistic equation, and our model fitted the experimental data from BNU-China well. Furthermore, we adjusted the RBS properties to make a sensitivity analysis (Figure 3.). The result is that our Kill Switch obtained a more effective reaction in a smaller temperature range. (Click here to see the model of Kill Switch.)
Experimental Plan
If we have extra time in the wet lab, we will characterize our Kill Switches according to the following protocol.
- Plasmids construction and transformation: Insert DNA fragments of BBa_K3606027 and BBa_K3606028 ordered from IDT into pSB1C3. Transform the two kinds of constructed plasmids into DH5α strain as experimental groups and empty pSB1C plasmids as control group. Culture three groups in 60mL LB medium (with 50 ng/µl ampicillin) at 37℃ overnight.
- Cold treatment: Divide each group into two test tubes for 30℃-culture groups and 37℃-culture groups. (3 for each temperature).
- Measure growth situation: Extract 5μl bacteria solution from each test tube every 1h. Diluted each bacteria solution to 10^7 times and culture them on three LB plate (with 50 ng/µl ampicillin) at 37℃ for 24h. Count the number of colonies in 5 cm^2 per plate after cultured for 24h at 37℃.
- Draw the growth curve to see the growth difference between the three group.
In this page we mainly explained our design of cold triggered toxin/antitoxin Kill Switches from their necessity, design consideration and demonstration, displaying that we had a reasonable and preeminent design of Kill Switch.
Reference
[1]Whitford CM, Dymek S, Kerkhoff D, et al. Auxotrophy to Xeno-DNA: an exploration of combinatorial mechanisms for a high-fidelity biosafety system for synthetic biology applications. J Biol Eng. 2018;12:13. Published 2018 Aug 14. doi:10.1186/s13036-018-0105-8 [2] Ronneau S, Helaine S. Clarifying the Link between Toxin-Antitoxin Modules and Bacterial Persistence. J Mol Biol. 2019 Aug 23;431(18):3462-3471. doi: 10.1016/j.jmb.2019.03.019. Epub 2019 Mar 23. PMID: 30914294. [3] Jones, Brian V. “The human gut mobile metagenome: a metazoan perspective.” Gut microbes vol. 1,6 (2010): 415-31. doi:10.4161/gmic.1.6.14087 [4] Carbonic Anhydrase and Bone Resorption, Nutrition Reviews, Volume 31, Issue 3, March 1973, Pages 101–102, https://doi.org/10.1111/j.1753-4887.1973.tb06005.x [5] Yamaguchi, Y., Inouye, M. Regulation of growth and death in Escherichia coli by toxin–antitoxin systems. Nat Rev Microbiol 9, 779–790 (2011). https://doi.org/10.1038/nrmicro2651 [6] Unterholzner, Simon J et al. “Toxin-antitoxin systems: Biology, identification, and application.” Mobile genetic elements vol. 3,5 (2013): e26219. doi:10.4161/mge.26219
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