Team:UCAS-China/Safety

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• safety
• Cell-free system
• SAH absorption
• Temperature-sensitive suicide switch
• Reference

Cell-free system

Cell-free system is a new field in synthetic biology. Specifically, in cell-free projects, components within the reaction system are produced and extracted by synthetic or engineered bacteria and later assembled to function in the same system without the need to introduce any genetically modified living organisms into the system. It will allow us to know in advance what substances may be produced in the reaction system and ensure that it does not pose a threat to the human body.

This is particularly important for environmental, medical or food projects, where leakage and contamination of engineered bacteria are most likely to have serious consequences. In fact, a number of previous iGEM projects have taken note of this and used cell-free systems, and some of them, like us, have used enzyme immobilization processes.

According to article, cell-free systems have several advantages over traditional methods: first, they cannot replicate and spread, reducing the risk of biological contamination; second, they do not have the same reduced efficiency as living cells that must allocate energy to metabolic processes; furthermore, their reduced complexity makes it easier to modify, for example, in our project, we could adapt the system to high-temperature coffee through directed evolution environment.

Some views have raised concerns about the efficiency of enzymatic reactions in cell-free reaction systems. We believe that the enzyme immobilization system we use can address this issue, as enzyme immobilization is a relatively mature bioengineering technology and has been used in industry, such as in juice production. Therefore, we believe that the efficiency of the reaction to remove caffeine from our cartridge can be ensured if we pay attention to the immobilization process and ensure that it does not seriously affect the enzyme activity.

SAH absorption

S-Adenosyl-L-Homocysteine(SAH) is formed by the demethylation of S-Adenosyl-Methionine(SAM)inthereaction.However, SAH is slightly harmful to human body.SAM is the most important methyl donor for various cell methylation reactions, and SAH has a inhibitory effect on SAM-dependent methyl transferases, thus affecting cell metabolism. High serum SAH concentration is considered to be a risk factor for cardiovascular complications in patients with chronic kidney disease.

We want to be able to adsorb this small molecule so that it does not escape into the coffee environment and enter the user's digestive system.

Therefore, we need a material that can absorb SAH firmly in large quantities to prevent the leakage of SAH absorbed last time. After comparison and analysis, we choose Tulsion® ADS-600, a kind of amino acid adsorption resin.This is a Moist spherical polystyrene resin that can be easily put in and taken out.It has strong adsorption capacity for amino acids and is harmless to human body.Since that both SAH and SAM are amino acids, the use of this material may lead to the loss of SAM, but considering the higher affinity between CkTcS and SAM, this problem has little impact under the condition of sufficient SAM.

After communicating with the distributor Kehaisi company, we learned that this amino acid adsorption resin is produced by Tulsion Company in the United States, and is widely used in food and drug production industry. Data kept by Kehaisi indicates that the resin performed well in a weak acidic solution similar to coffee.

Temperature-sensitive suicide switch

Although following the planned process, our engineered bacteria will not cause safety problems, we have added an insurance policy - a temperature-sensitive suicide switch - to prevent possible accidents and leaks in the lab.

We designed a temperature-sensitive suicide switch using toxin RelB and antitoxin RelE, and added it to the upstream of the two enzyme’s genes . In a scene of industrial production, as long as the temperature is down to below 35 degrees Celsius, the engineered bacteria will suicide immediately to avoid microbial leakage and contamination.

In the meantime, we would like to follow another temperature-sensitive suicide switch that UCAS-China involved in last year's project. In the gene circuit, CI434ts as a transcription factor can repress TEVts and doc toxin expression, while TEVts as a protease can cut CI434ts, thus forming a balanced relationship. And when the temperature decreases, CI434ts activity decreases and loses its inhibitory effect on downstream toxins, thus initiating the engineered bacteria suicide program.

To improve the performance of this suicide switch, we also fused a hybrid tevS/ssrA tag to the C-terminal structural domain of Doc toxin to reduce the leakage expression of Doc toxin. For more information on this part, see our project last year

Reference

P. M. Ueland. (1982). Pharmacological and biochemical aspects of S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase. Pharmacological reviews, 34(3), 223-253. doi:0031-6997/82/3403-0223

T. Sheahan, H. J. Wieden. (2021). Emerging regulatory challenges of next-generation synthetic biology. Biochemistry and Cell Biology. doi:10.1139/BCB-2021-0340

Ou, S., Tang, X., Li, Y., Liu, Q., Zhang, F., (2013). Relation ship between serum S-adenosylhomocysteine level and cardiovascular complications in patients with chronic kidney disease. Chongqing medical journal, 42(6), 613-615. doi:10.3969/j.issn.1671-8348.2013.06.005