For us, the safety of our group, the people around us and the environment are the topmost priority. Therefore, we made sure to protect ourselves and others by following all safety guidelines given by the Gentechnikgesetz (GenTG) and the Gentechnik-Sicherheitsverordnung (GenTSV) in Germany as well as the European Laws regarding biological lab work. With our work, we hope to make our waters safer and for this the following safety rules have been in place.
Before being allowed to work in the laboratory, we had an introduction by the responsible professor.
During this introduction, we learned all rules of lab work as well as the safety measures we have to
follow in an S1-laboratory. We got introductions to all equipment and potential dangers. All of us
worked in the necessary protective gear, consisting of a lab coat and, depending on the experiment,
gloves and glasses. The S1-introduction was mandatory for the permission to work in the lab at all. The
introduction also included the measures taken regarding waste disposal in the lab, both for non
genetically modified organisms (GMOs) and everything that came into contact with GMOs.
At the beginning of the project, we held a bootcamp for those members of the team that had limited or no
prior experience in the laboratory. It consisted of basic techniques, such as pipetting and platting, as
well as more specific techniques necessary for the project, such as prepping plasmids and transforming
microorganisms. The more experienced students taught the others regarding these techniques. There was
also an introduction in some of the equipment of the lab in addition to the specific equipment from the
work groups. Additionally, the safe work in the lab, as introduced during the S1-instruction, was taught
and shown.
The organisms used during our experiments are Escherichia coli strains. We used only strains that are
not pathogenic and usable within S1-laboratory conditions. The strains used are the BL21 DE3 and Top10.
They are good for transformations and expression of proteins. We ensured that all modified organisms
were removed from the lab following the GMO-regulations. The cells were given to us from the work groups
from the institutes of Biochemistry and Molecular Biosciences. The modifications with our
GoldenGate-plasmids are not a danger to the environment due to the cells not being exposed directly to
the environment. Furthermore, the reporter proteins are GFP and beta-galactosidase, both of which are
not dangerous and are degraded fast in the cells.
Antibiotics
For both the selection after the transformation, in the pre- and main cultures as well as the testing of
our alginate capsules (Bobas) and our paper strips, we used antibiotics. Due to kanamycin, ampicillin
and the other antibiotics we used being acutely toxic and having hazardous effects on the human body, we
ensured that everybody on the team knew how to handle, store and dispose them.
Reagents
For our gels, we have used acrylamide and gel colouring substances. These solutions are toxic and
mutagenic and, therefore, should be handled with great care. Acrylamide is a necessary component for
SDS-gels and is only to be used while wearing gloves and protective glasses. The colouring for the gels
can have its effects upon contact or digestion, therefore, the solutions are handled with gloves,
protective glasses and care.
All other reagents have also been handled according to the instructions given by the producer and with
great care.
Paper Strips
During the production of the Bobas themselves, there is little risk for both us and our environment as
the alginate-cell-mixture is only a problem when not disposed of correctly. During the cell selection
process and the testing of the functionality and the necessary concentrations, we used antibiotics. The
cell selection used the antibiotics as a selection factor in the medium. For the functionality tests, we
used antibiotics in varying concentrations to find the limits of our system.
Bobas
For the cell selection, we again used antibiotics as a selection factor. During the creation of the
paper strips themselves, no dangerous reagents were used. However, for the testing solutions various
amounts of antibiotics were used to define a range of functionality. The paper strips used were all
disposed of following the GMO-regulations.
Proof-of-concept
For both parts of the project, we did short proof-of-concept experiments without the
Two-Component-systems on the GoldenGate-plasmids. We instead used plasmids with inducible GFP or
beta-galactosidase for a proof-of-concept beforehand. The methods were the same as for the finished
Bobas and paper strips. The protein-production is induced with IPTG or made evaluable with X-Gal in a
wide range of concentrations. This is both for a test of functionality as well as a potential standard
curve.