The iGEM Team Tuebingen respects and complies with all safety and security rules set by the iGEM competition.
Laboratory Practice
Before working in the lab (safety level 1), all members of the iGEM Team Tuebingen have received detailed biosafety and security introductions on general lab practice, handling of hazardous material and behaviour in emergencies. Additionally, all avoidable risks in the practical work were substituted (e.g., usage of Midori Green instead of Ethidium Bromide). Laboratory work was conducted according to Sarah Schulz’s Good Lab Practice guideline (see Downloads) and was under faculty supervision at any time.
All organisms used in course of the laboratory work were non-pathogenic. E. coli cells of the DH5-Alpha strain, Novablue™ cells (E. coli K-12) and E. coli Nissle 1917 are standard organisms for various cloning experiments and were developed for laboratory applications.
Project Design
The aim of the project is creating a probiotic for the therapy of Type 2 Diabetes Mellitus. The chosen organism E. coli Nissle 1917 is non-pathogenic, isolated from human feces. It has been applied in probiotic therapies since the early 1920s and is considered to be a safe biological carrier for human use [1].
In the delivery of the therapeutic substance, the incretin mimetic Exendin-4, from the intestine to the pancreas, a cell penetrating peptide (Penetratin) is involved. It is considered to be a safe transmucosal delivery vector, since tests showed no unwanted co-absorption [2] and no irreversible effect on the intestinal epithelial membrane [3]. Additionally, we consulted biosafety officers Dr. Jörg M. Schibl and Dr. Brigitte Walderich as well as our advisor Dr. Bastian Molitor, concluding the cell penetrating peptides chosen for this project are safe to use (safety level 1) (Human Practices/Experts).
To implement a biocontainment mechanism in E. coli Nissle 1917, we are using the CRISPR/Cas3 system. It is controlled by three conditions ensuring the viability of the bacterium is limited to the human intestine, avoiding the spread of GMOs in the environment. For more information see (Project/Kill Switch).
References
- Westendorf, A., Gunzer, F., Deppenmeier, S., Tapadar, D., Hunger, J. K., Schmidt, M. Buer, J., Bruder, D. “Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules.” FEMS Immunology & Medical Microbiology, Volume 43, Issue 3, March 2005, Pages 373–384.
- Khafagy, E., Iwamae, R., Kamei, N., Takeda-Morishita, M. “Region-Dependent Role of Cell-Penetrating Peptides in Insulin Absorption Across the Rat Small Intestinal Membane.” NCBI, 11/17/2015.
- Kristensen, M., Nielsen, H. “Cell-penetrating peptides as tools to enhance non-injectable delivery of pharmaceuticals.” NCBI, 04/18/2016.
