In our project we want to establish a new production platform for biofuel production based on ‘artificial endosymbiosis’. We choose two of the most used and well established model organisms in genetic engineering: Saccharomyces cerevisiae and Escherichia coli. By following and extending the guidelines for responsible and safe research, we are able to ensure minimal risks to experimenter and environment. Here, you can find all important safety aspects we followed through the course of this summer. Additionally, we focused a whole side project on safety - we created a complete database of all kill switches ever designed within the iGEM competition and were able to model four of them in detail.

Before starting our lab work, all students received a detailed safety instruction by covering all important aspects of working safely. This instruction was held by the safety officer of the Max Planck Institute for terrestrial microbiology Prof. Dr. Brandis-Heep, as well as Volker Vincon. A lab specific safety instruction was also given by our instructors. This covered for example:

• proper behaviour in the lab, S1 safety measures and practices
• documentation of experimental work
• use of personal protective equipment
• waste disposal of biological samples
• chemical handling and hazards
• accident prevention measures
• fire safety regulations and emergency behaviour

Additionally, each student received an in depth training in every facility and for every instrument we had to work in or with (e.g. confocal microscope, FACS, HPLC) and was supervised during their measurements.

Since our project is meant to work as an industrial scale production, we wanted to keep safety issues as minimal as possible. Especially concerning our ‘endosymbiosis’ approach, we rejected several ideas for safety reasons. In the beginning of our project we wanted to use invasin and listeriolysin as an invasion method for our symbionts - this mechanism originates from Yersinia pestis and Yersinia tuberculosis and is part of their virulence factors, which makes them BSL3 and BSL2 organisms, respectively. Therefore, we choose a different and passive approach, a polyethylene glycol (PEG)-mediated fusion of vesicle enclosed microorganisms with yeast spheroplasts, which does not involve any pathogenic factors and therefore is harmless for humans and environment. During the lab work only BSL1 organisms have been handled, such as different strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Escherichia coli.

In Germany there are several important laws and regulations that provide all information about the handling with chemicals and biological samples. The so called Gentechnikgesetz, a law regarding regulations in genetical engineering, covers all regulations we mentioned in the ‘Lab Safety’ part. Additionally, there is the Biostoffverordnung, which covers personal protection when working with biological samples and several chemicals. A detailed list of all organisms and their risk assessments can be found in the Organismenliste.

We think safety, especially concerning biocontainment, is an issue all of us need to assess. Instead of designing our own kill switch, we took advantage of the numerous kill switches already implemented in the iGEM database. We were creating a model describing their escape probability in terms of evolutionary stability understanding them as a genetic network. We did so in collaboration with the iGEM team of Lethbridge, who performed experimental work in order to verify our model. In addition to the modeling of several kill switches, we included all kill switches ever mentioned in iGEM since its beginning in 2004 in a database. This collection is available for download here. Hopefully this can help many teams to gain insight in already existing kill switches, their stability and how to improve the already available kill switches.

For the sending of our parts, we used the provided DNA Submission Kit and followed the instructions coming with it.