Safety

Used organisms

In our project, we used Escherichia coli and Bacillus subtilis to create a system to produce and secrete a variety of enzymes that are known to aid in the separation of ink particles from paper fibers. A process known as deinking. While the assembly of the enzyme expression constructs was performed in E. coli, the enzyme expression and secretion is done in B. subtilis. To this end we designed a shuttle backbone which allows for propagation in both E. coli and B. subtilis. The secretion is facilitated by either of the leader peptides nprb and sacB, which are N-terminally fused to the gene of interest as part of a "tag". These tags consist of a promotor, a RBS and the nprb or sacB sequence. We planned to use 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.

E. coli as well as B. subtilis can be handled at biosafety level one. Additionally, B. subtilis is Generally recognized as safe (GRAS) by the American Food and Drug Administration.

The E. coli strain used is the commercially available "NEB Turbo" strain. The B. subtilis strains utilized by us are the commercially available strain ATCC 6051 and the strain LS8P-D, a strain optimized for protein expression and lacks several proteases to improve protein yield. This strain was provided to us by the group of Prof. Schweder at the Institute of Pharmacy, department of Pharmaceutical Biotechnology from the University of Greifswald.

It has the following genotype: ΔsacA::SpecR, ΔlytC::lox72, Δbpr-spo::lox72, ΔnprB::lox72, Δmpr::lox72, ΔaprE::lox72, ΔnprE::lox72, Δvpr::lox72, Δepr::lox72, ΔwprA::lox72

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 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.

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.