一句短语概括整个页面，不要用overview这类无实际意义的词或词组
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Engineering of Kill Switch
添加一个概述
Brainstorm
To be responsible for the environment, we considered it is necessary to introduce a Kill Switch to deprive of the survivability of engineered E. coli outside human bodies. Kill Switch is a gene device to eliminate bacteria in a short period of time when engineered bacteria escape from the set conditions. In the context of our project, the set condition is the human intestine and the condition under which we wanted our bacteria to die is the outside environment. Therefore, we first come up with a distinguishing condition in the intestine and in nature – glucose concentration. Glucose concentration is higher than the environment and is a very general condition that may enable the Kill Switch to apply to many situations.
Tentative design
After searching literature, we learned several special glucose-sensitive promoters could facilitate a different state of expression in the two conditions. We found two glucose-sensitive promoters in the part registry ( rpoH P5 and PT-αCRP ). For example, PT-αCRP can turn on downstream genes precisely and autonomously in response to glucose limitation, and for this reason, it is also called glucose-starvation promoter. So, we imagined our Kill Switch to be a glucose-starvation promoter followed by a toxic protein gene.
Consultant
We further researched the parameters of the two promoters to forecast the efficiency of the Kill Switch. For one thing, the trigger concentration of them is both 0.01-0.05%, while the glucosuria level of diabetics and pregnant women can be far higher than that[1]. However, our target consumer is the elderly who also often have diabetes, glucose-starvation promoter may not be activated effectively in their excrement. For the other thing, the model of rpoH P5 made by BNU-China demonstrated that glucose concentration in the intestine would drop under o.o5% within 2 hours after meals, which impeded persistent calcium supplement function[2]. For more practical information, we consulted the designer of rpoH P5 from Beijing Normal University. He didn’t consider glucose concentration as a specific signal to trigger elimination outside the body and also recommended us try toxin/antitoxin systems to enable more flexible regulation. (Click here to see our Integrated Human Practices page for detailed information.)
Further Research, Consultant and Redesign
After consultant, we decided to undergo an overhaul of our Kill Switch. Therefore, we traversed most suicide circuits in intestinal probiotics by iGEM teams and the response systems and the killer systems summarized they used. We cataloged biobricks of the killer system and the sponsor system separately and figure out the fittest part in the two groups to compose our Kill Switch. (Click here to see the two tables.)It consists of RelE/RelB toxin-antitoxin system and RNA thermometer NoChill 06 to regulate it.
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.
Coincidentally, we found BNU-China has created a similar design in 2019. We had another consultant with BNU-China, they approved our design. This year, we had very limited time in wet lab, so we decided to collaborate with them, obtaining their wet lab experimental data to construct model and verify our design. We improved the logistic equation, and our model fitted the experimental data from BNU-China well. Furthermore, we adjusted the RBS properties to make an efficiency analysis. The result is that our Kill Switch obtained stronger reactions in a smaller temperature range (Figure 1). (Click here to see the model of Kill Switch .)(Click here to see the model of Kill Switch.)
And we ordered DNA fragments from IDT in the case that we have enough time to test our Kill Switch design in wet lab. Our experimental plans to measure the growth curve of two Kill Switch were as follows:
- Plasmids construction and transformation: Insert DNA fragments of BBa_K3036004 and BBa_K3606027 ordered from IDT in to pSB1C3. Transform the two kinds of constructed plasmids into DH5α strain as experimental groups and empty pSB1C plasmids as a control group. Culture three groups in 60 mL 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 5 μL bacteria solution from each test tube every 6 hours. Diluted each bacteria solution to 10^7 times and culture them on three LB plates (with 50 ng/ μL ampicillin) at 37℃ for 24 hours. Count the number of colonies in 5 cm^2 per plate after cultured for 24 hours at 37℃.
- Draw the growth curve.
Learn and Improve
To find some detailed parameter to constructing a model of Kill Switch, we found literature that we didn’t notice before[3]. From this literature, we found RelE has several characteristics that need to be considered with caution. First of all, RelE is a very rare toxin that has the activity against both prokaryotic and eukaryotic organisms in a similar mechanism. It has been observed that RelE can induce apoptosis in cultured human cell lines. Besides, RelE/RelB module tends to have unexpected enrichment in bacteria that is not observed for other toxin/antitoxin modules such as MazE/MazF and ParD/ParE. Such enrichment may be associated with the formation of the persistence of microbes in the gut. In addition, the divisions of bacteria carrying homologs of the RelE toxin involved in comprising the human gut microbiota. Though all these effects only been observed in artificial mammalian gene expression systems, whether it has a tangible impact on the human gut microbiome has not been verified yet, we should be particularly careful with the safety issues.
So, we decided to replace the RelE/RelB system with another toxin/antitoxin system. MazF/MazE, an efficient and commonly used toxin/antitoxin module mentioned in that literature, shows no evidence of unexpected enrichment and a health threat to human. Therefore, MazF/MazE may perform better in intestinal Kill Switch. In our minds, every little improvement involving safety is a huge issue. We additionally ordered a new DNA fragment (BBa_K3606028) from IDT, hoping to characterize it following the protocol above in the future.
Reference
[1] Cowart SL, Stachura ME. Glucosuria. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Chapter 139. Available from: https://www.ncbi.nlm.nih.gov/books/NBK245/ [2] https://2019.igem.org/Team:BNU-China/Model#Glucose [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
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再来例子 Market Analysis
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