Safety and Security

1. Would any of your project ideas raise safety issues in terms of:

1.1 Researcher Safety?

At the beginning of our project we attended a general health & safety induction and were given a safety tour of our lab involving guidance in waste disposal of sharps, trace chemicals, and biohazardous material. Labcoats and disposable gloves were worn at all times in the lab and removed upon exiting the lab to ensure that any contamination could not spread outside of the lab. Good laboratory practice, such as regular hand-washing and frequent cleaning of the workbench, was enforced. Safe Operating Procedures (SOPs) for both general safety and for equipment used in our project were closely followed at all times. While working in the lab, we were supervised by our instructors, advisors or lab technicians from the university’s School of Life Sciences Learning & Teaching staff.

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.

1.2 Public Safety

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.

1.3 Environmental Safety

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.

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.

2. Do any of the new BioBricks parts (or devices) that you made this year raise any safety issues?

No.

3. Is there a local biosafety group, committee, or review board at your institution?

Yes. Comprehensive risk assessments must be carried out prior to the start of the project. Any accidents or spillages of micro-organisms must be reported. Correct disposal of waste in accordance with University regulations. The University of Dundee Biological Health and Safety Management Policy can be viewed here

We discussed the details of our project with both the Health & Safety Coordinator and Information Officer for our College of Life Sciences. They helped us to produce a risk assessment specific to our project. This GMO risk assessment was approved by our College of Life Sciences Biological Safety Committee.

We were given a general lab safety induction which included guidance in waste disposal of biohazardous material. Documents describing Standard Operating Procedures and risk assessments were made available to us. We also received informal training in various protocols including miniprep, gel extraction, PCR and cell transformation.

Genetic Modification Legislation from the Scottish Government can be seen here.
Biosafety guidance from the Health & Safety Executive can be found here.
Guidance from the Health & Safety Executive specifically relating to GMOs can be found here.
The Cartagena Protocol on Biosafety

4. Do we have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

Our team has created a Safety Database called “The Syn Bin”. Based on a system in use at our university which has proved successful, we have created a database to store records of accidents that members of iGEM teams have experienced. Teams can then learn from the experiences of others and potentially avoid having similar accidents.

The Syn Bin can be found here