1. General safety

Genetically Modified Organisms (GMO)

Cell chassis

To standardize and document biosafety, the World Health Organization made classification standards for organisms in four ‘Risk Groups’, and safety precautions in four ‘Bio-Safety Levels’. These standards can be found in the [http://www.who.int/csr/resources/publications/biosafety/en/Biosafety7.pdf WHO Laboratory biosafety manual].

The only organisms used for this project are Escherichia coli strains ‘DH5-Alpha’, ‘Mach1’, ‘BL-21’ and ‘JM109’. They are all non-pathogenic laboratory strains. The likelihood of a human getting ill from working with these bacteria is therefore low. The most probable route of transmission would occur by accidental aerosol formation or ingestion. The Environmental Protection Agency states that the K-12 strain (which both DH5-Alpha and JM109 are derivatives of) is poorly retained in the human gut, so the chance of it becoming pathogenic by mutations is low[1]. Mainly for these reasons, this organism is classified as Bio-Safety Level 1. Although we couldn’t find any official records about safety aspects of E. coli B (BL-21) and W (Mach1) strains, both are common laboratory strains that are commercially available. Literature suggests that both these strains can be handled at the same Biosafety level[2-4]. The Wageningen UR laboratory treats these strains at BS Level 1, and so have we.

GMO in general

There are several scenarios in which unintentional release of genetically modified material could take place. Labeling of the lab equipment and glassware used is necessary to prevent loss and improper waste disposal by a fellow researcher. The following problems are not as easy to prevent.

The air filtering system will have a hard time in keeping aerosols in the lab when a window gets broken. If this happens through a thunderstorm, an electricity break-down is not unlikely and would increase the chance of release furthermore. Under Good Microbial Practice, though, the formation of aerosols is prevented as much as possible, thus the total probability hereof is rather low.

All waste containing genetically modified organisms is required to be sterilized by autoclaving before disposal. Direct actions should be taken if it is discovered that an autoclave has been malfunctioning after the waste is discarded outside of the lab. To reduce the risk on environmental contamination it is necessary to check upon the autoclave’s functionality (by monitoring its operational temperature). If the aforementioned hazards do occur, they should be reported to the Minister of ‘Housing, Spatial Planning and the Environment’ and involved institutions to make the hazards undone as soon as possible.

Cell chassis enhancement

The use of antibiotic resistance markers increases the chance of spreading antibiotic resistance to pathogens. By conjugation, transduction or natural genetic transformation, DNA can be transferred between bacteria. There is a chance the antibiotic resistance genes end up in a pathogenic bacterium which is not intrinsically resistant to antibiotics. However, the chance that these genes persist is low, since there is no direct evolutionary benefit for micro-organisms living around the lab to take up the extraneous genes.

We have used plasmids that bear resistance markers for Ampicillin, Chloramphenicol, Kanamycin and Septinomycin. These are all resistance markers that are used in BSL-1 laboratories on a standard basis.

GMO with our BioBrick System

In the event of (un)intentional release of a bacterium containing our BioBrick system, the physical conditions outside the lab are harsh to the E. coli strain chassis that are used in this project so the bacterium would be unable to grow. Still, by means of natural genetic transformation, the DNA of the BioBrick system could be taken up by other bacteria that are better suited for the conditions.

The Wageningen 2012 iGEM project is about Virus-Like Particles (VLPs) armed with a leucine zipper for standardized attachment of ligands or structures. The Coat Protein monomers that make up these VLPs have no known activity besides forming a cage and thus raise no foreseeable safety issues. [More...]

The leucine zippers bind strongly to their opposite parts and also (with less affinity) amongst themselves. Although they might be able to bind to different, natural proteins as well, this does not raise any foreseeable safety issues.

Expression of the biobrick plasmid would give a host organism no foreseeable evolutionary advantage without the artificial antibiotics selection pressure, but instead incur a significant metabolic burden. It is therefore considered unlikely that the plasmid would be propagated. Unfortunately it is not possible to know in advance what the actual effect of a natural transformation would be, but the odds are in favor of it to cause little impact on the environment.

Risks and benefits

Under Good Microbiological Practices the risk of working with the BioBrick system is rather small to the researcher. In itself, the cell chassis that is used is not considered to be dangerous to human beings. Despite the involved BioBrick system of our project, the chassis that we use is not pathogenic or harmful. Even if the system was to be released into the surroundings, there is a low risk that it would enter a pathogenic host organism. However, there might be some risk involved if a person or organization has malicious intents by using this system as a delivery gateway for toxins. In this case they would be able to arm our VLPs with a toxin and possibly even make it infect only specific species or cell lines.

We believe, though, that every new tool that is made could be used for malicious purposes, but by no means does this mean they shouldn’t be made. In our case, we think that the possible benefits of our system and the likelihood of it being used for those purposes outweigh the risk of it being used for evil purposes.

References

1. [http://epa.gov/biotech_rule/pubs/fra/fra004.htm Final risk assessment of Escherichia coli K-12 Derivatives]

2. Chart, H., et al., An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha and EQ1. J Appl Microbiol, 2000. 89(6): p. 1048-58.

3. Studier, F.W., et al., Understanding the Differences between Genome Sequences of Escherichia coli B Strains REL606 and BL21(DE3) and Comparison of the E. coli B and K-12 Genomes. J Mol Biol, 2009. 394(4): p. 653-680.

4. Park, J.H., et al., Escherichia coli W as a new platform strain for the enhanced production of L-Valine by systems metabolic engineering. Biotechnology and Bioengineering, 2011. 108(5): p. 1140-1147.