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. (2015) 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.

We asked people questions about biosafety in a survey (Street science survey) that we conducted in downtown Bielefeld. The questions included, if they preferred a sensor with or without living microorganisms. The results showed that people preferred a cell-free sensor. That points out the public awareness when it comes to microbiology and genetically modified organisms.

Safety check of our approaches

Safety check of the plasmid repressor interaction assay (PRIA)

At first glance, our two approaches the 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 be compared to ELISA, an assay which is broadly used for example in hospitals (Lequin 2005), in terms of biosafety. 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 the environment.

We want to do 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 the 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 comprises an antibiotic resistance which can be avoided through cloning of DNA sites without the use of antibiotics. An appropriate method is e.g. cloning with antibiotic-free selection by using auxotrophic strains (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 addressed properly.

For further assessments of PRIA's biosafety status we interviewed Dr. Mathias Keller from the district government in Detmold ("Bezirksregierung Detmold"). He is the responsible officer for biosafety aspects and supervision of labs working with genetically modified organisms (GMOs) in East Westphalia-Lippe region where Bielefeld is located (District government Detmold). We asked how the district government deals with the release of DNA from genetically-modified organisms into the environment. We were wondering if this release may cause any legal concerns. According to Dr. Keller the release of pure DNA is not governed by the German Genetic Engineering Act. This is true 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 unequivocally 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 environment where a close 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 requirements 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 one does 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 than cell-based systems.

Safety check of the Cell-Free Protein Synthesis (CFPS) approach

How can we separate the advantages of E. coli from the biosafety concerns?

We use the best features from E. coli and get rid of the unnecessary ones! We disrupt the cells, thereby disabling the bacteria to reproduce, and we collect the cell extract with its molecular machinery to produce proteins in vitro, that is without living cells!

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 the cell debris are separated from the molecular machinery needed for CPFS (see CFPS protocols). Although the cell disruption is very efficient (we had a maximum of 8 colony forming units in 100 µL of final cell extract), there are still 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 the California Institute of Technology explains: "[...] we have seen both cases where colony forming units come out of the extract and when they don't." "[...] but 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 would not be occurring in the CFPS environment. In contrast, cell-based biosensors throughout have to deal with several issues. One of those is for example the problem that GMOs are brought outside of the laboratory. However, none of these problems occur when the system is cell-free. Recently, methods to ensure that ones CFPS setup is really cell-free have been described by Smith et al. 2015. We investigated these methods within our project. You can find the results here at the CFPS result page.

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 building a completely cell-free biosensor. It is very likely that our final application passes every existing biosafety test regulated by current law. 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

Biosafety, ok. And Biosecurity? Read about Dual Use

Our first biosensor design: CFPS