Safety

Used organisms

In our project, we engineered B. subtilis bacteria to produce and secrete a variety of enzymes that are known to facilitate the separation of ink particles from paper fibers, a process known as deinking. Secretion is induced through one of two tag constructs called nprb and sacB. The tags consist of a promotor, RBS and sequence to induce secretion optimized for B. subtilis. We used the produced supernatant to deink paper in our small-scale deinking setup to determine the deinking efficiency of our expressed enzymes and compare them to conventional chemical deinking. To ease cloning, we first assembled all of our enzyme expression constructs in E.coli and then transformed them into B. subtilis for protein expression. To do this we have used a specially designed hybrid backbone which allows for the propagation in both E. coli and B. subtilis.

The B. subtilis organism we were using is a specially engineered strain called LS8P-D which has following genotype: ΔsacA::SpecR, ΔlytC::lox72, Δbpr-spo::lox72, ΔnprB::lox72, Δmpr::lox72, ΔaprE::lox72, ΔnprE::lox72, Δvpr::lox72, Δepr::lox72, ΔwprA::lox72

The strain is optimized for protein expression and as a results lacks several proteases to improve protein yields. The strain was given to us by the group of Prof. Schweder at the Institute of Pharmacy, department of Pharmaceutical Biotechnology from the University of Greifswald.

The E. coli strain we were using is “NEB Turbo”.

Risks

Our project poses the standard risks associated with biotechnological work. We work with S1 organisms which are nonpathogenic and do not cause disease in healthy humans. 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 during all experiments conducted and all waste or equipment that has come into contact with bacteria is sterilized.

Actions

As our project is designed to be scaled up for an application at an industrial level, the main risk associated with our project is the unintentional release of genetically modified organisms into the environment with our produced recycled paper. To eliminate this risk either the supernatant used for deinking would need to be sterilized before deinking or the paper itself after deinking. Future work to asses these risks would involve testing different sterilization techniques and analyzing them for their applicability to industrial large scale processes. The main methods considered at the moment are filtration of the supernatant using a rotational filtration system or gamma irradiation of the paper itself.