Our project engineered the peroxide repressor PerR to act as a switch for the evolution of defenses against oxygen in obligate anaerobe Clostridium tyrobutyricum, and made the transformation of it from an obligate anaerobe into a facultative anaerobe.

In the real world, when our project could be further developed, it can realize the growth of several obligate anaerobic microorganisms for soli modification and other effects which required the engineering bacteria to survive both anaerobic and aerobic conditions, to support various space experiments, expand the experimental categories, provide new ideas and new conditions for astronauts' space research, and help interstellar research.

If possible, our technique may help more duodenum and ileum patients. Since the intestinal environment was often considered anaerobic and the duodenum and pylorus are partially microaerobic environment [1], and C. tyrobutyricum has been shown to act as a probiotic, we expected that the engineered bacteria can exert its therapeutic role under microaerobic conditions.

Studies have proved the effects of C. tyrobutyricum in mice on protecting intestinal morphology [2,3]. We also hope that it can contribute to the treatment of human gastrointestinal diseases.

The metabolites of C. tyrobutyricum can improve the soil fertility, increase the yield of crops. After submitting the experimental results to the safety testing department, and synthesizing lots of experimental data to ensure its safety, the soil transformation could be carried out in the test field to observe the effect on crop cultivation, and then test the safety of crops and promote them to planters around the world after confirming safety.

We could also provide companies with less costly and efficient fermentation methods that do not require the creation of a rigorous anaerobic environment to complete the fermentation and manufactory process towards obligate anaerobes with multifunction, such as Clostridium butyricum producing butyric acid or just be produced as feed additives ^[3], under conditions with a small amount of oxygen or even air exposure.

Genetically modified C. tyrobutyricum in the laboratory may be leaked out of the laboratory into the external environment, causing resistance genes(like chloramphenicol resistance) transfer, affecting the growth of wild-type bacteria, but it is clear that C. tyrobutyricum is not harmful to people as a species of probiotics^[2,4,5].

There is no doubt that the suicide switch, which is one of the biosecurity measures recommended by iGEM, is a very effective safety method, and it is also what we have always wanted to design^[6]. However, constructing such a device in the gram-positive bacterium C. tyrobutyricum is difficult and needs to be explored.

In the future, we also hope to continue to explore methods such as suicide switches to ensure the safety of genetically modified C. tyrobutyricum , together with other gram-positive bacteria to the world.

We always understand that the real world is always more complex than we know. From the transformation of soil in outer space, the relationship between intestinal flora and disease treatment, or the production of probiotic products, there are much more mechanisms stay unknown and worth exploring.

There must be many retrofit sites that we haven't found yet to really develop our project into a perfect system and application Perhaps these retrofits are more effective in achieving the goal, and the existing solutions may be not cheap and effective enough to be easily implemented into real world.

[1] Kalantar-Zadeh, K., Berean, K.J., Burgell, R.E. et al. Intestinal gases: influence on gut disorders and the role of dietary manipulations. Nat Rev Gastroenterol Hepatol 16, 733–747 (2019).

[2] Xiao Z, Liu L, Jin Y, Pei X, Sun W, Wang M. A Potential Prophylactic Probiotic for Inflammatory Bowel Disease: The Overall Investigation of Clostridium tyrobutyricum ATCC25755 Attenuates LPS-Induced Inflammation via Regulating Intestinal Immune Cells. Mol Nutr Food Res. 2021 Jul;65(14):e2001213. doi: 10.1002/mnfr.202001213. Epub 2021 Jun 9. PMID: 34021704.

[3] Wang Zongwei, et al. Research Progress on Mechanism of Clostridium butyricum Affecting Intestinal Barrier and Its Application in Livestock and Poultry Breeding. Chinese Journal of Animal Nutrition VL 34 IS 6.

[4] Xiao Z, Liu L, Pei X, Sun W, Jin Y, Yang S-T and Wang M (2021) A Potential Probiotic for Diarrhea: Clostridium tyrobutyricum Protects Against LPS-Induced Epithelial Dysfunction via IL-22 Produced By Th17 Cells in the Ileum. Front. Immunol. 12:758227.

[5] T Hudcovic, et al. Protective effect of Clostridium tyrobutyricum in acute dextran sodium sulphate-induced colitis: differential regulation of tumour necrosis factor-α and interleukin-18 in BALB/c and severe combined immunodeficiency mice. Clinical and Experimental Immunology, Volume 167, Issue 2, February 2012, Pages 356–365.

[6] Whitford, C.M., 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 12, 13 (2018)