Biosafety issues are main aspects of iGEM projects (Guan et al. 2005) and play a major role in our project. One of our main aims this summer was to develop applications that are completely cell-free. We were inspired by Pardee et al. who – by creating an ebola sensor – showed how cell free synthetic biology can be a medium to transfer the findings and ideas from the lab to the field.

In a survey (Street science survey) that we conducted we asked people questions about biosafety, for example if they preferred a sensor with or without living microorganisms. The results showed that people preferred a sensor without living microorganisms. That points out the public awareness when it comes to microbiology and genetically modified organisms.

Safety check of our approaches

At first glance, our two approaches PRIA and CFPS differ when it comes to biosafety concerns. PRIA, as an assay-like approach that utilizes purified proteins and DNA, involves no living organism. PRIA is an in vitro method and can (in terms of biosafety) be compared to ELISA, an assay which is broadly used for example in hospitals (Lequin 2005). Nevertheless, recombinant DNA is used in this assay. Although plasmid DNA persists only to a minor degree in host cells (Moe-Behrens et al. 2013), DNA is a molecule that bacteria from the environment can coincidentally absorb (Cérémonie 2004) and that therefore can affect environment.

We can imagine a risk assessment to determine to which extent DNA is released in the environment when using PRIA. In any case, we would recommend the user to destroy the test strip after usage, for example by high temperatures, to degenerate DNA (Nielsen et al. 2007). It is also possible to modify the DNA that is used: For example it is conceivable that it includes a kill-switch or comparable safeguards (Moe-Behrens et al. 2013). Another problem is that the used DNA owns an antibiotica resistence which can be avoided through cloning of DNA sites without antibiotica. An appropriate method is e.g. cloning with antibiotic-free selection through alanine (iGEM Team Bielefeld 2014). To conclude, we think that in future applications PRIA can be used outside of the lab under the condition that these concerns are adressed properly.

For further assessments of PRIA's biosafety status we interviewed Dr. Mathias Keller from the district government in Detmold ("Bezirksregierung Detmold") where he is the responsible officer for biosafety aspects and supervision of labs working with genetically modified organisms (GMOs) of the region East Westphalia-Lippe where Bielefeld is located (Bezirkungsregierung Detmold). We asked how the district government deals with the release of DNA of genetically-modified organisms into the environment if this release may cause any legal concerns. According to Dr. Keller the release of pure DNA does not come under the German Genetic Engineering Act no matter how the DNA is produced. The Act just deals with the handling of GMOs. Therefore, it is important that there are no GMOs in the product. Considering biosafety aspects it has to be unequicovally proven that the purified protein extract and plasmid DNA preparation are cell-free.

Moreover, the release of DNA happens permanently. So to which extent is the biosafety affected? According to Dr. Keller, the uptake of DNA depends on the uptake frequency, the encoded genes (e.g. conferring antibiotic resistance or encoding toxins) and the selection advantage for the transformed cells. He lists examples for the environments where an intimate contact between DNA and cells are likely to occur, like sewage treatment plants, our digestive tract and the soil. In his opinion, the risk that possibly released DNA from our system is incorporated by competent cells and that these cells proliferate and spread out is so low and unlikely that it is negligible. Furthermore, we asked him which advantages and disadvantages he sees in cell-free systems. He replied that no approval requerements in regard to German or European genetic engineering regulations are required for cell-free systems so that they are universally usable. For the use of these systems you do not need to be a user of a genetic engineering facility. Biologically/chemically he sees as advantages that the stability, durability and insensibility against external impacts in cell-free systems are considerably higher than in cellular systems. Moreover, he finds cell-free systems as more suitable for miniaturization. In conclusion, our expert assured us that cell-free systems are safer and better to use for the user.

Our second approach CFPS utilizes cell extract for transcription and translation. Cell extract is generated by cell disruption, for example via sonification, followed by centrifugation steps in which cell debris is divided from the molecular machinery needed for CPFS (see CFPS protocols). Although very efficient (we had a maximum of 8 colony forming units in 100 µL of final cell extract), still there are some cells left after this procedure.

We wondered if it was possible to create a biosensor based on cell extract that is completely cell free. We contacted experts in the field of cell free biology and asked them about this topic. Mr. Zachary Z. Sun, PhD candidate at California Institute of Technology, explains: "[W]e have seen both cases where colony forming units come out of the extract and when they don't." "[B]ut if one is careful [one] can get no cells", says Dr. Michael Jewett from Northwestern University. Regarding biosafety, it would be a clear advantage if reproduction is not occurring in CFPS environment. In contrast, cell-based biosensors throughout have to deal with several issues. These are for example the problem that GMOs are brought outside of the laboratory. However, none of these problems occur when the system is cell-free. If one uses only a bulk of purified components, like the PURE system does (Shimizu and Ueda 2010), the risk of living cells in the sensor are negligible.

For our final application, we applied cell extract and all compounds needed for protein synthesis on paper and lyophilized it. We proved that absolutely no E. coli survives this process by placing the paper onto an LB plate and putting it into a 37 °C incubator for 2 days. We could not observe any colony forming units, not after any performance of extract lyophilization. This is consistent with recent findings from Smith et al. 2015 who showed that sterile filtration and lyophilization are methods to free cell extract from any cell left. Lyophilisation was preferred due to stable expression thereafter, which fits to our results.

Safety check of our final application

A paper-based test strip for the use in the field has many advantages, like we depicted throughout our project. A smartphone camera to measure fluorescence within a cheap and simple device offers great potential. Apart from the molecular mechanisms that create or weaken fluorescence, we revealed no further disadvantages.

In conclusion we established two approaches for the building of a completely cell-free biosensor. It is very likely that our final application passes every existing biosafety test. With this project we provide ideas and results that are useful for future iGEM projects, especially regarding biosafety. Projects based on cell free approaches will probably benefit to a great extent from our findings and results. We consider this a great contribution to the iGEM community.

References

Ceremonie, H.; Buret, F.; Simonet, P.; Vogel, T. M. (2004): Isolation of Lightning-Competent Soil Bacteria. In: Applied and Environmental Microbiology 70 (10), S. 6342–6346. DOI: 10.1128/AEM.70.10.6342-6346.2004.

Guan, Zheng-jun; Schmidt, Markus; Pei, Lei; Wei, Wei; Ma, Ke-ping (2013): Biosafety Considerations of Synthetic Biology in the International Genetically Engineered Machine (iGEM) Competition. In: BioScience 63 (1), S. 25–34. DOI: 10.1525/bio.2013.63.1.7.

Lequin, Rudolf M. (2005): Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). In Clinical chemistry 51 (12), pp. 2415–2418. DOI: 10.1373/clinchem.2005.051532.

Moe-Behrens, Gerd H G; Davis, Rene; Haynes, Karmella A. (2013): Preparing synthetic biology for the world. In: Frontiers in microbiology 4, S. 5. DOI: 10.3389/fmicb.2013.00005

Nielsen, Kaare M.; Johnsen, Pål J.; Bensasson, Douda; Daffonchio, Daniele (2007): Release and persistence of extracellular DNA in the environment. In: Environmental biosafety research 6 (1-2), S. 37–53. DOI: 10.1051/ebr:2007031

Smith, Mark Thomas; Bennett, Anthony M.; Hunt, Jeremy M.; Bundy, Bradley C. (2015): Creating a completely "cell-free" system for protein synthesis. In Biotechnology progress. DOI: 10.1002/btpr.2157.

What we built with our Biobricks