In our project, we worked with the organisms Escherichia coli and Streptococcus mutans.
The E. coli strain used for the cloning of our genes of interest the commercially available "NEB Turbo" strain. (https://www.neb.com/products/c2984-neb-turbo-competent-e-coli-high-efficiency) It has the following genotype: F ́ proA+B+ lacIq ∆ lacZ M15 / fhuA2 ∆(lac-proAB) glnV gal R(zgb-210::Tn10)TetS endA1 thi-1 ∆(hsdS-mcrB)5.
For protein expression, we used the BL21 strain, which we kindly obtained from the Laboratory of Natural Product Chemistry, Dr. Jeroen S. Dickschat, Bonn. This strain is also commercially available. (https://www.neb.com/products/c2530-bl21-competent-e-coli) The genotype of this strain is: fhuA2[lon] ompT gal [dcm] ∆hsdS.
They both underlie the biosafety level S1.
To investigate the biofilm formation of the cariogenic bacterium S. mutans on saliva-coated polystyrene plates, we used the S. mutans strain ATCC 25175 that we kindly obtained from the Institut für Pharmazeutische Mikrobiologie, Prof. Dr. Tanja Schneider, Bonn. This strain is also commercially available. (https://www.lgcstandards-atcc.org/products/all/25175.aspx?geo_country=de) In the U.S.A., this organism underlies the biosafety level 1 as defined by the U.S. Public Health Service Guidelines. However, in Germany this organism underlies the biosafety level 2 which is why we were only allowed to work with it under the supervision of trained personel in the Institut für Pharmazeutische Mikrobiologie, Prof. Dr. Tanja Schneider, Bonn. We did not genetically modify this organism.
Our project poses the standard risks associated with biotechnological work. For the genetic engineering, we work with S1 organisms which are non-pathogenic and do not cause disease in healthy humans. The two proteins that we planned to express by E. coli are not toxic. The Glycosyltransferase-SI hydrolyses sucrose into fructose and glucose and builds water-soluble and water-insoluble glucans from the glucose. It may also bind these glucans. The antigen I/II is a surface protein that binds to pellicle proteins from human saliva bound to the surface of teeth. Both genes for these proteins derive from S. mutans and were synthezised by IDT. All our work is conducted in a biosafety level 1 laboratory. The main risk associated with our project is the unintentional release of genetically modified organisms into the environment or the contact of team members or others in the lab with the genetically modified organisms. Actions taken to reduce these risks are standard safety level 1 procedures: access to our laboratories is limited to instructed and trained personal, eating and drinking are prohibited in all lab areas, the use of lab coat and gloves as well as proper laboratory clothing (i.e. long pants and solid shoes) is mandatory at all times and all waste that has come into contact with bacteria is sterilized by autoclaving. Surfaces and Equipment are disinfected with Kohrsolin extra, one of the Robert-Koch- Institute (RKI) approved disinfectants.
All the work with S. mutans was conducted in a biosafety level 2 laboratory under the supervision of trained personal in the Institut für Pharmazeutische Mikrobiologie, Prof. Dr. Tanja Schneider, Bonn. We strictly adhered to the rules of an S2 laboratory.
As our project is intended to be of therapeutic use, our aptamers would first have to be tested in vivo in pre-clinical animal studies and in clinical trials to assess their safety and to test them for any side effects.
Bulk quantities of the aptamers would be chemically synthesized under well controlled conditions.