Safety

General Safety

Safety is a big issue, not only in biological laboratories but also pretty much in any research facility worldwide. This is why our universities in Graz and in Manchester established general safety rules that are mandatory for all researchers in our facilities. Food and cosmetic material is absolutely forbidden inside the lab.
When we enter the lab we put on lab coats and wash our hands to make sure to not bring any contamination into our lab. When working with living organisms we make sure to wear gloves and safety goggles must be used when working with harmful substances.
Using ethidiumbromide is only allowed in specially designated rooms and special, nitrile gloves have to be worn at all times. Also no equipment that might be contaminated with ethidiumbromide can be taken out of that room and we must not take any general labmaterial, such as lab books, computers or even Eppi racks into that room.
Special UV proof gloves have to be used when visualizing stained DNA under UV light.
When handling hot material such as autoclaved glass wears or media compositions, heat proof gloves must be worn.
Certain items of equipment that we are not familiar with require an introduction to us by a more experienced member of the lab.
When working with delicate materials that need special protection against contamination not only Bunsen burners but also sometimes the lamina flow hoods are used.
For all researchers it should be self explanatory that you wash your hands every time you leave the lab!

Safe project design

The quorum sensing systems used in our expression system come from potential pathogens. Pantoea stewartii subsp. stewartii which originally contains the EsaR/I system is a plant pathogen causing Stewart’s wilt, a serious disease of corn. Burkholderia cenocepacia, delivering the CepI/R system, is an opportunistic pathogen for humans with cystic fibrosis or chronic granulomatous disease. So to avoid working with these strains we got the genes synthesized rather than obtaining them from the bacteria via PCR.
The enzymes used for the synthetic pathways of L-DOPA and Dopamine should not pose any safety risks. They are of eukaryotic origin and impose a heavy load on E. coli, so it is highly unlikely for them to spread by giving a selectional advantage.
For the first stages only standard laboratory strains of E. coli (BL 21 and TOP10) are used. Additionally the used ampicillin resistance ensures a minimal viability of the used plasmids outside the ampicillin media in our lab. The same applies to the integration of our construct into the probiotic E. coli Nissle 1917. The (at this point highly theoretical) integration of this modified probiotic strain into patients’ gut floras however would require a whole lot of other biosafety considerations, we don’t have to fully consider yet. However, the setup of our multidimensional expression system allows for the introduction of a suicide mechanism at a high specific bacterial density to reduce the chance of overgrowth of the natural gut flora.

Safe lab work

Outside the usual “good lab practice” no other safety measures were necessary for our work.

Safe shipment

We sent in our BioBricks through the standard shipping process, required by iGEM. In addition we sent our pCERI vector to the iGEM Team Norwich by spotting it on steril filter paper, according Rosman, Miller. Improved method for plasmid shipment. Biotechniques. 1990 May;8(5):509. This way there was no need to send living organisms.