Safety is the primary and prolonged demand for our project. In our whole experimental process, we mainly have three major aspects of safety considerations, including safety of our project design, safety during our experiment, and future plans.
Safety of project design
The probiotic platform that our team aims to develop is expected to colonize in human’s gut.
Because one of our goals is to develop a long-lasting probiotic platform, we need to make our bacteria successfully colonize the gut for a long time. First, we choose E. coli Nissle 1917 as final chassis since it is a homogeneous strain within our intestine, which does not cause immune response. This way, our engineered bacteria will not interfere too much with existing microbiome. Additionally, in order to prevent exclusion of our engineered bacteria, we aim to enhance their ability to compete with local microbes by promoting their proliferation. We wish to employ the strategy of overexpressing glucose dehydrogenase (GDH), an enzyme responsive to the pentose phosphate pathway. This enzyme has been tested by BNU-China that it confers a significant proliferative advantage, and chosen by them as way to promote colonization.
As a probiotic platform used human gut, we decide that we should ensure that people could have an easy way to control its function. We hope that the users could stop the platform function whenever they want. As a result, we decide to apply an MazF gene after a L-arabinose promoter into our bacteria. According to BNU-China 2019, L-arabinose doesn’t significantly present in regular diet, and it doesn’t have side effects for human uptake (BNU-China, 2019). As a result, L-arabinose could serve as an adequate molecule to induce MazF expression, which could lead to transcription inhibition, leading to the death of the engineered bacteria. This pathway is also not harmful for human gut microecosystem because it does not lead to cell lysis.
Figure 1. Suicide induction design. Created by biorender.com.
To further ensure its safety even after it is excreted outside gut by chance, our team designs a cold-inducible switch that aims to ensure that the E. coli will kill itself when it leaves human (BNDS-China, 2023). Because there is a large difference between the human gut temperature, which is about 37 degrees Celsius, is largely different to the environment. When this change happens, our cold-inducible switch will be activated. At gut temperatures, the two proteins, TEV ts-18 and TF ts-2, inhibit each other in the system. However when the temperature reaches the environment temperature, TEV proteins will degrade the TF protein, which normally also inhibits an endolysin that is responsible for bacteria suicide. As a result, with exposure to environment temperature, E. coli will commit self-lysis.
Figure 2. Cold-inducible switch design. Created by biorender.com.
Safety during experiment
During our project we conducted most of our experiment in a BSL-1 laboratory. During the whole process of our experiment all of our members wear lab coats and gloves. For any experimental processes involving bacteria, we conduct them in a biosafety cabinet. We are also aware for nearby butane burners.
Before conducting our experiment, our team members have experienced long-term training on lab safety, including proper equipment usage, disposal of chemicals, and response to emergency situations. Also, we are taught by instructors of detailed experiment procedures.
In our laboratory, we sterilize the biosafety cabinet using UV light. During our experiment, we have some methods to prevent potential UV light harm to us. A blue light transilluminator is utilized when visualizing agarose gel, which is relatively safe while we are also wearing proper facial protection.
Certain antibiotics are utilized during the experiment to select transformants. To eliminate the possible side effects to human, our team conducted a survey before the project, so that we ensure that none of our members are allergic to them. Antibiotics storage and use are also under appropriate security protocols.
Future plans
In order to fulfil our group’s final goal of marketing our project, the Clinical trails consent guidelines should be followed. A Clinical Trail Authorization(CTA) will be gained for clinical trails from Phase I to Phase III. Manufacturing permission of active pharmaceutical ingredients(AIPs) as well as our whole product would be obtained, following the Drug Admission Law(DAL).