Chemical Safety

As a risk reduction measure we opted to use Qiagen kits rather than phenol based protocols. Ethidium bromide is an intercalating agent (inserts into the DNA helix) in common use in laboratories as a means of detecting nucleic acids through agarose gel electrophoresis. As ethidium bromide distorts the structure of the DNA helix, it is a mutagen and carcinogen. To avoid the risk of exposure to ethidium bromide, we decided instead to use GelRed stain in our agarose gels.

Biosafety

Escherichia coli is a Gram-negative bacterium which is naturally found in the colon of warm-blooded organisms including humans. While some strains of E. coli can cause serious food poisoning in humans, most are harmless. The bacteria Salmonella enterica is a Gram-negative bacterium that often infects poultry and cattle and can result in salmonellosis. Portrayal of these particular bacteria by the media has greatly influenced public opinion and therefore it is important that we ensure our strains are completely harmless and that accidental release would not endanger researchers or members of the public.

We used a number of different bacterial strains throughout our project. E. coli MG1655, JM110 and DH5α [10] are disabled, non-pathogenic, non-toxicogenic, non-colonising, laboratory-adapted K12 strains [11], which are widely used for research purposes and present no hazard to human health. We also cloned genes from Salmonella enterica serovar Typhimurium LT2a strain, which is severely attenuated making it non-pathogenic [12]. Our project did not involve culture of Salmonella but began with genomic DNA extraction from a Salmonella cell pellet. Neither the individual structural proteins nor the assembled microcompartment itself are known to have any harmful properties.

These strains are allowed to be handled as class 1 pathogens rather than class 2 so are not categorized as a biohazard. Although these strains are non-pathogenic, it is still important to take measures to prevent contamination. Any protocols involving the transfer of bacterial cells or colonies between plates or tubes, were carried out in sterile conditions – close to a Bunsen flame. Otherwise, bacterial cells remained in lidded dishes or universal tubes with the caps tightly sealed. All biological waste was disposed of according to lab waste disposal protocols, which involves autoclaving biological waste prior to discarding it.

b. Risks to the safety and health of the general public if released by design or accident?

While carrying out our project, utmost care was taken to ensure that no biological or chemical materials were released from the lab. However, if accidental release were to occur, the E. coli K12 strain would pose very little or no danger to human health due to its poor ability to colonize the gut and establish infections. E. coli K12 also appears to lack the ability to produce significant quantities of toxins that affect humans [11]. Neither the individual Pdu proteins that we will express in E. coli K12 nor the assembled microcompartment will alter the host cells in any way that will make them more hazardous to human health.

We are using ampicillin resistant genes within our plasmid as a selectable marker for bacterial transformations. As we are aware of the issues surrounding horizontal gene transfer and multi-drug resistant bacteria, we are following university protocols regarding GMO waste disposal. The ultimate goal of our project, as with any iGEM project, is for our modified bacteria to be used practically in the environment. Our final step would be to remove antibiotic resistance from our plasmid prior to release of our bacteria into the environment.

c. Risks to environmental quality if released by design or accident?

E. coli K12 has been shown to survive poorly in the environment, has a history of safe use and is very unlikely to have adverse effects on animals, microorganisms or plants. Since E. coli is not a normal inhabitant of soil, it would not be expected to survive under these conditions. Studies have shown that introduction of E. coli K12 into non-sterile soil in saline resulted in a decrease to levels below detection after 21 days. E. coli K12 cannot survive well in seawater and would be expected to quickly decline in air due to drying conditions and low levels of nutrients. It also has no known survival mechanisms, for example it lacks the ability to produce spores. Thus, as E. coli K12 survives poorly in soil, air and water, its survival in the environment would be severely limited [11]. Finally, the genes encoding BMCs are frequently laterally transferred in nature and already widespread among the bacterial phyla.

d. Risks to security through malicious misuse by individuals, groups or states? Specifically, are any parts or devices in our project associated with (or known to cause): - pathogenicity, infectivity, or toxicity?

E. coli K12 is non-pathogenic and not capable of colonising the gut, thus cannot cause infection and cannot produce dangerous toxins in significant quantities. 1,2-propanediol degradation is associated with growth of Salmonella in host tissues [13]. However this is likely to be a consequence of the degradation pathway itself rather than the BMC shell, which has merely a structural role and acts only to contain the pathway. Our empty BMC shell contains no proteins capable of degrading 1,2-propanediol [7] and so should not enhance the survival characteristics of the E. coli K12 host.

- threats to environmental quality?

As mentioned previously, any E. coli K12 released into the environment would be expected to survive poorly and would not prove harmful to plants, microorganisms or other animals. As BMCs are already widespread among the bacterial phyla, lateral transfer of our BMC would be unlikely to cause harm.

- security concerns?

Due to the non-pathogenic, non-toxicogenic, and non-colonising nature of E. coli K12 and the harmless nature of our parts, we do not foresee any security concerns with our project. Our laboratory has secure entry to prevent unauthorised access.