Team:Hamburg/Safe Design

Safe Design

In our project we worked with genetically modified organisms (§3 para. 2 GenTG) and encountered many levels of safety risk. But we always acted accordingly to the respective danger and to the legal requirements.

Risk and safety assessment

n order to be able to work according to the correct safety guidelines, we first carried out a risk assessment according to §5 GenTSV and Attachment I GenTSV. According to this, cell lines and E. coli DH5α are generally RG1, whereas K. pneumoniae and P. aeruginosa are RG2 (see BVL 78/2009/4). So we needed to work under S1 and S2 conditions (§7 GenTSV, §7 GenTG).

Safety briefings

Every member oft he team got a S1 and S2 safety briefing according to §12 para.2+3 GenTSV, §12 para. 1 Arbeitsschutzgesetz(Labor Protection Law) and GUV-R A1(Principles of prevention). In this briefings, amongst other things, we learned about the labeling and handling of dangerous and/or infectious materials, Rules of conduct in the laboratory and the waste disposal.

Of course we’ve always worn appropriate protective clothes and equipment Attachment III GenTSV), as for example lab coat and gloves as well as glasses if working with CMR substances like ethidiumbromide.

When leaving the laboratory, we made sure that every organism we worked with got/would get inactivated according to §3 para. 5 and §13 para.3+4 GenTSV for not contaminating anything outside the lab. Another safety measure to prevent the release of a GMO were deadman switches that should prevent the survival of our GMOs outside the labs.

Klebsiella pneumoniae

To synthesize yersiniabactin / nor-pyochelin, we used irp1, irp2, irp3, irp5, irp6, irp7 and fyuA from K. pneumoniae, which are in nature important for synthesizing yersiniabactin in these bacteria. For this, irp 2-7 and fyuA were synthesized, whereas irp1 was acquired from a life strain of K. pneumoniae. As mentioned above, we needed to do this in special S2 labs and under special safety conditions according to Attachment III para. 2 GenTSV and §7 GenTG. Because siderophores are iron scavenging peptides and thus help bacteria to survive in iron deficient surroundings, it is an essential virulence factor during infections to overcome basic host defense mechanisms as reducing free iron is one the first countermeasures to combat infections. But since we won’t induce the whole pathway, there is no additional safety risk. However, it might be that under substrate conditions the test-bacteria are able to derive siderophores and increase their proliferation. For these upcoming problems we included many more safety measures, for example splitting up the genetic code for transport and production and put them onto different plasmids. So, to utilize yersiniabactin four plasmids need to be present in the same organism plus the salicylate pathway, what makes this problem unlikely to occur.

Pseudomonas aeruginosa

To verify the possible merits of pyochelin as an antimicrobial agent loaded with cytotoxic ions, we used pchcd pchefg and fptA from P. aeruginosa. Naturally, these proteins synthesize the siderophore Pyochelin as well as a transport protein to ship it through the membrane and are not listed in the Australia Group List or the U.S. Select Agents and Toxins List. We obtained the proteins via DNA synthesis and because of our bacteria lacking the whole pathway and thus be unable to synthesize the siderophore at will, there’s no additional safety risk, even Pyochelin is a virulence factor created to scavenge iron-ions to boost proliferation in bacteria.

Since P. aeruginosa is an Organism of RG2, the same increased safety measures as with K. pneumoniae apply.

Toxicology studies

We used K. pneumoniae substrain pneumoniae carrying yersiniabactin genes and P. aeruginosa substrain pao1 for toxicological experiments. For this purpose, we cultured these human pathogens in a separated S2 lab, which was provided by our second affiliated research group, Prof. Dr. Peter Heisig at the University of Hamburg and handled them only under S2 safety workbenches.

What we do for safety

In the beginning and in the end of our project, we spoke with Mirko Himmel (Integrated HP) about our safety standards. In addition, Professor Heisig and his wife are the safety representatives for S2, and they are supervising us every day in the lab.