Overview

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Our team developed a novel kill switch composed of gene suppression tools and toxic genes, and combined this kill switch with substance detection to create a new type of whole-cell biosensor as a test paper. In the future, on the one hand, we will associate our project with the mobile APP to produce a more convenient and efficient whole cell test paper detection system. On the other hand, we will develop a series of new kill switches and organize a kill switch toolkit that can freely control the survival time of engineered bacteria. Thereby, we can protect the safety of human and nature, while protecting the more stable development and application of synthetic biology technology.

Kill switch

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Our project attempts to construct epigenetic-modified kill switches in both yeast and E.coli, and microbes equipped with such kill switches are called "Wind-up cells."

In yeast, we first tried to construct the kill switch by combining dCas9 with the deacetylase SIR2, and then we planned to try to use a stronger epigenetic modification tool HML sequence to construct a more efficient kill switch. After inserting the toxic gene between HML-L and HML-E sequences, the experiment showed that the expression of the toxic gene was well inhibited and existed in engineering bacteria for a long time. In follow-up experiments, we are trying to further transform the HML sequence into a convenient and efficient kill switch

In E. coli, we first tried to construct the kill switch by binding the promoter of mioC to DAM methylase, and the experimental results showed that this design may produce a certain inhibition effect on the toxic gene. Then, we investigated and used CRISPRi to build the kill switch, this design has also been shown to inhibit toxic genes. In the future, we plan to combine methylase with CRIPSPRi to construct fusion proteins, and try to construct more effective kill switch in E. coli than the above two.

Figure 1. The design of kill switch

Test paper

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We combined the apparent modification-based kill switch with substance detection to create a safer whole-cell biosensor as a test paper for detection. We are now testing the effectiveness of our test paper by designing genetic circuits to enable these cells to detect tetracycline and isopropyl thiogalactoside. When we need to use this test paper, The Wind-up cell's kill switch is activated immediately after leaving the culture environment, starting the countdown to cell death. At this time, the substance to be detected (tetracycline and IPTG) induces the expression of downstream genes, so that cells can express a large number of fluorescent proteins, and we determine whether there is a substance to be detected by fluorescence detection. After the detection function is completed, the countdown of cell death ends, and the engineered microorganisms with Wind-up Cell system on the test paper will inevitably die.

Figure 2. The design of test paper

Application

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In the future, on the one hand, we will further improve our kill switch and associate the fluorescence detection signal with the mobile APP, which will not only improve the detection intensity of the fluorescence signal, but also facilitate the reading and analysis of the detection results, and produce a more convenient and efficient whole-cell test paper detection system.

On the other hand, our project will replace different toxic genes and combine them with various epigenetic modification tools to develop a new series of kill switches and organize a kill switch toolkit that can freely regulate the survival time of engineered bacteria. This toolkit can be adapted to various application scenarios of engineered bacteria, is expected to reduce the application risk of engineered bacteria and clear the way for new synthetic biology technologies to be put into practice, thus bringing revolutionary changes to the development of synthetic biology technology.

When our toolkit is applied to environmental treatment, different switch combinations can be selected for different pollutants and application scenarios, thus eliminating the risk of biological treatment of environmental pollution, so that engineered bacteria can be allowed to enter the pollution and express related enzymes such as AlkB, AlmA and metallothionein in the countdown of life. Sufficient degradation or adsorption of pollutants. To solve the oil pollution, pesticide pollution, heavy metal pollution, and other major ecological problems, and return the earth to green mountain and clear water.

Our toolkit can make engineered bacterial therapy more accurate and safe, bringing it one step closer to clinical application. When combined with drug delivery in vivo, we can select the appropriate switch to set the appropriate drug release time according to the needs of different diseases, to prevent the excessive release of drugs and the generation of drug-resistant bacteria. When combined with tumor therapy, the kill switch toolkit can make the engineered bacteria produce different treatment durations and intensities for different tumor types such as solid tumor and metastatic tumor, and at the same time, it can die after targeted tumor removal to prevent its killing of normal cells around the tumor. In addition, when applied to the diagnostic imaging phase, the engineered bacteria can die after targeted enrichment of the imaging agent, which facilitates the safe metabolism of the imaging agent.

When our kill switch kit is applied to the production of natural compounds, the engineered bacteria can only survive during the corresponding fermentation period and will die in time after the fermentation, eliminating the ecological risk caused by the spread of engineered bacteria in the environment and promoting the rapid development of biological production.

Figure 3. Associate with the mobile phone APP

Figure 4. Kill switch toolkit

Significance

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Our project can significantly reduce the risk of the spread of genetically engineered microorganisms in the environment so that engineered microorganisms create value in all aspects while reducing the risk of environmental safety. Therefore, with the rapid development of biotechnology in the future, social and ecological security can be more effectively protected. At the same time, our project enables synthetic biology technology to better accept the management of biosafety regulations such as the United Nations Cartagena Protocol on Biosafety, the European Union's Regulations on the Environmental Release of Genetically Engineered Microorganisms, and China's Measures for the Safety Management of Genetic Engineering^[1]. While reducing biosafety risks, it also reduces people's concerns about synthetic biology technology, which is helpful for conserving the safe and stable development of synthetic biology technology.