During the midterm of our project research, we had already successfully addressed chassis safety concerns. However, there were still some pressing safety and experimental issues that required immediate attention. Therefore, we made the decision to collaborate with teams from other universities in order to seek solutions and gain valuable insights.
As a result, several members of our team actively participated in the CCiC conference held in Suzhou, Jiangsu in July. During this conference, we engaged in extensive discussions with project teams from renowned universities such as Wuhan University and China Pharmaceutical University. The primary focus of these communications and discussions revolved around crucial topics including probiotics' colonization mechanisms within the intestine and their potential for dislodgement, any potential disruptions caused by probiotics on intestinal microbiota balance, ensuring the true safety of probiotic suicide switches, as well as addressing environmental concerns following their expulsion from the body.
The meeting provided our team with detailed suggestions on the challenges we encountered, offered a wider range of possibilities for the colonization and decolonization plans of this project, and furnished us with a preliminary understanding of colonizing and decolonizing the intestines. This knowledge is invaluable for further design and research in this field.
1.Zhejiang Shaoxing Institute of Tianjin University
1. In terms of intestinal colonization, the research team from Zhejiang Shaoxing Institute of Tianjin University has proposed four ideas:
① We can induce bacteria to express specific adhesins on their surface, while the intestinal wall contains a mucus layer with targeted molecules. By selecting molecules that can bind to these adhesins, we can achieve precise intestinal colonization.
② Utilizing flagella as a platform, we can display proteins on the bacterial surface to enhance their adhesive capabilities.
③ Physical materials can be employed for encapsulating small molecules that adhere to the intestine.
④ Protein interactions involving binding antigens and antibodies also offer potential for achieving targeting effects.
2. Regarding dislodging colonization, the team suggests incorporating a linker into the displayed protein on the bacterial surface. Through specific hydrolytic enzyme cleavage of this linker, we are able to effectively dislodge colonization and ensure that bacteria do not persist in the intestine causing harm.
3. Concerning disruption of intestinal microbiota balance, the team is utilizing Escherichia coli Nissle 1917 and Lactococcus lactis F44 in their study on intestinal microbiota and assures minimal possibility of disturbing its equilibrium. They are currently conducting mouse experiments and will provide feedback on experimental results in due course.
2.China Pharmaceutical University
(1)Regarding the selection of chassis cells: The bifidobacteria utilized by this research team are naturally occurring in the human gut and do not disrupt the balance of the intestinal microbiota; in fact, they can even enhance this equilibrium. Research has demonstrated that bifidobacteria possess anti-inflammatory properties within the intestine. They exhibit tolerance and adhesion capabilities within the gastrointestinal tract.
(2)Regarding intestinal colonization: Bifidobacteria, as chassis cells, possess inherent ability to adhere to the intestine, thereby achieving successful intestinal colonization.
(3)Regarding dislodging colonization: The team indicated that while exerting therapeutic effects, bacteria employed in their studies will also accumulate a toxin in vivo. Once this toxin reaches a certain threshold level, bacteria will undergo automatic cell death, resulting in their shedding from the intestine. Therefore, they will not persist within the body for an extended duration.
(4)Regarding potential environmental contamination: The project is designed with two suicide switches - one responsive to oxygen concentration and another sensitive to temperature changes. Upon expulsion from the body or if there is accidental spillage of culture medium, bacteria will perish due to alterations in oxygen levels or temperature (Note: The temperature switch is still under development).
3.Wuhan University
(1)Quorum Sensing (QS Safety System): The research team has identified that the QS safety system can regulate the dynamic equilibrium of gut microbiota, leading to automatic elimination upon expulsion from the body.
(2)Regarding colonization displacement: This issue remains unresolved as its life cycle is unknown, uncertain and uncontrollable even though it can be expelled from the body.
4.Nanjing Zhuoqingtang Education Technology Limited Liability Company
(1)On Gut Colonization: EcN itself is an inherent gut microbiota capable of long-term residence and reproduction within intestines.
(2)Concerning disruption of gut microbiota balance: They have developed an EcN concentration sensing mechanism which triggers cell oligomerization when strain concentration becomes too high; this leads to growth and reproduction until a certain amount is reached, at which point oligomerization switch reactivates to reduce concentration for achieving dynamic balance. However, specific details require experimental validation and feedback data for determination.
(3)Whether It Will Contaminate Environment After Expulsion: Modified EcN strain has much larger load compared to normal strains resulting in very low survival competitiveness making it unable to survive.
Summary
Valuable suggestions were provided by teams from Wuhan University and China Pharmaceutical University regarding issues such as gut colonization, dislodging colonization, and controlling biological leakage during our experimental process. These suggestions not only enriched our repertoire of relevant strategies but also offered preliminary insights into gut colonization and dislodging. This has significant guiding implications for the subsequent design and research of engineered bacteria, aiding us in achieving our project goal of "treating diseases when they occur and enhancing health when there are none." In terms of addressing the disruption of gut microbiota balance, Wuhan University's QS safety system serves as an important point of reference for us. This system provides crucial insights into maintaining the dynamic equilibrium of gut microbiota. In the future, we may also attempt to construct a similar system and validate the effects of Zymomonas mobilis on the dynamic balance of gut microbiota through modeling and other methodologies.