Team:Humboldt Berlin/Safety
Based on the WHO - Laboratory biosafety manual, 4th edition1, the factors associated with the highest likelihood of incidents occurring are: activities associated with aerosolization, activities associated with sharp material and low competency of personnel. To prevent any of these incidents from occurring, every team member working in the laboratory was briefed in biosafety level 1 (BSL1) standard. Everyone had to comply with the lab guidelines at every time. All lab work was carried out in a BSL1 laboratory provided by Prof. Dr. Erhardt. Genetically engineered organisms were protocoled according to local guidelines. Experiments performed with organisms that require sterile ambients (Escherichia coli and Salmonella Typhimurium) were performed either at a clean bench or close to a burner. All used reusable lab consumables were properly autoclaved. For further information, see iGEM Safety Form.
Working with Salmonella Typhimurium is intrinsically dangerous as these pathogens are mostly known for causing disease that affects the intestinal tract. As our project goal clearly involves injection of given minicells into the patient, we had to carefully review options to decrease the danger our minicells could pose to patients. As a start, we created minicells from an attenuated strain of Salmonella Typhimurium, ST14028, which is deficient in SPI-2 and thus BSL 1.
However, we planned to overexpress SPI-1 as to increase the SPI-1 T3SS count on the minicell surface. This should, in theory, improve the secretion of our protein of interest. By doing so, we expect the virulence of the respective minicells to increase as well. To counter the potentially increased virulence, the minicells were planned to be attenuated. Several genetic modifications are known to reduce the virulence. We decided to delete galE. galE encodes for a galactose epimerase and is essential for the synthesis of the LPS and lacks a complete O- antigen. This decreases the survival of Salmonella typhimurium in macrophages2. Our aim was to reproduce those results and show that our attenuated minicell are characterized by a lowered virulence compared to
the wild-type.
List of Sources
Safety
General Safety in the Lab
Working with Salmonella
