Team:Dundee/Safety

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

Researcher safety
Three organisms were used in this project: Escherichia coli (E. coli), Salmonella typhimurium LT2a (S. typhimurium) (Swords et al. 1997) and Clostridium difficile (C. difficile). The genes for the Type VI Secretion System (T6SS) and Biosensor biobricks originated in S. typhimurium which were then cloned into the E. coli chassis. C. difficile was also handled during the project due to this being the targeted organism.

Numerous strains of E. coli were used throughout this project, all of which possess no threat to human health. E. coli DH5α, JM110 (cloning) and MG1655 (protein expression) were utilised all of which “are disabled, non-pathogenic, non-toxicogenic, non-colonising, laboratory-adapted K12 strains, which are widely used for research purposes” ( Dundee iGEM 2011 ). The E. coli strain K38 [pGP1-2], encoding resistance to Kanamycin was also utilised for the radio labelling experiments which was perfectly safe to handle and required no extra safety.
These E. coli strains are all classed as being Risk Group 1 per WHO guidelines ( WHO 2004 ). S. typhimurium is classed as a Risk Group 2 organism ( WHO 2004 )

The “non-toxic” strain of C. difficile, AY1 (Reynolds et al. 2011) was handled during cell growth experiments towards the final weeks of the project. All work carried out with C. difficile took place in the presence of an experienced researcher to ensure that correct safety procedures were adhered to such as the usage of a fume hood. According to EHO guidelines this organism is classed as level 2 organism ( WHO 2004 ).

The individual parts of the T6SS are believed to possess no harmful properties and thus no additional safety measures were carried out whilst handling these genes. The resulting biobricks are also considered to be safe for future handlers.

Some modifications were made to the original genes gained from Salmonella, these included site directed mutagenesis (SDM) in order to remove stop codons to fuse endolysin and mCherry to the ends of VgrG and Hcp. SDM was also used to remove unwanted restriction sites located within some of the genes. These modifications did not create a hazardous product and did not include the handling of dangerous chemicals.

35S-Methionine radiolabelling was carried out in order to characterise the combinatorial clones. Protein samples were prepared by an undergraduate member of the team but all practical work using the radioactive isotope was conducted by a highly experienced instructor who had clearance to do so. Handling of 35S-Methionine took place within a secure ‘hot room’, utilising the fume hood and Perspex shielding. Full safety protocols were followed at all times ( Amersham Biosciences. 2002 ).

The two plasmid vectors used in this project, pUNI-PROM and pSB1C3 were also safe to use and required no extra safety handling.
Endolysin from phage ΦCD27 was perfectly safe to use and handle. Endolysin proteins only affect bacterial cell walls and this endolysin specifically targets the cell wall of C. difficile so was not harmful to other organisms (Mayer et al. 2008).

Before beginning any practical work for the project, all members of the team attended a Health and Safety seminar and the divisional Lab Manager delivered a safety tour of the lab, providing us with essential information in order to complete a compulsory checklist ( CLS 2009 ). General laboratory safety was highly stressed to ensure the safety of ourselves and others within the lab. Documents describing Standard Operating Procedures and risk assessments were made available to us. Formal training in various protocols including miniprep, gel extraction, PCR and cell transformation was also provided.
Instructions were given on the correct disposal of waste materials ensuring the safety of lab technicians (e.g. from sharps) and enabling effective autoclaving and destruction of live organisms.
Basic safety procedures were used at all times by members of the wet lab team. Personal protective equipment (PPE) was worn at all times and upon leaving the lab, PPE was removed and hands thoroughly washed in order to prevent any potential contamination with the outside environment. Our group were closely supervised throughout the project by instructors or advisors and any poor techniques were quickly spotted and corrected.

Like that of the previous Dundee iGEM team ( Dundee iGEM 2011 ), Qiagen kits were used in place of phenol based protocols, which have been linked with long term affects such as cardiovascular disease ( US dept. H&S, Phenol. 2008 ).
In replacement of ethidium bromide, a well-known carcinogenic mutagen, the stain GelRed was utilised for our agarose gels which has been specifically designed by Biotium with safety in mind ( Biotium 2011 ).

The category two carcinogen and mutagen, Acrylamide, ( US dept. H&S, Phenol. 2008 ) was used on a daily basis in order to produce the protein gels utilised for Western blot analysis. Special care was stressed by the advisors and this was taken into consideration when using this compound.

Public safety
If accidental release of our modified E. coli (vector biobrick pSB1C3) were to happen, the organism would pose no more threat than the unmodified version. The Biobrick should be suitably expressed in any of the E. coli K12 strains mentioned above, which, as previously mentioned lack “the ability to produce significant quantities of toxins that affect humans” ( Dundee iGEM 2011 ). The addition of the Biobrick should not improve the organism in any way that would make the strain hazardous to human health.

Like any bacterial transformation, resistance to an antibiotic is required to decrease the chance of contamination with other bacteria. The pUNI-PROM vector provides resistance to ampicillin, whilst pSB1C3 has chloramphenicol resistance. To ensure that this would not pose much threat to public safety, all GMO disposal guidelines were followed. Washing of hands and the removal of lab coats also greatly decreased the chance of its release.
The transfer of multi-drug resistance, however, is a growing concern. As it is hoped that this project could be further developed and put to practical use in the future, a concluding task of the project could be to remove this resistance in order to put the public’s mind at ease.

Through the addition of a biosensor, expression of our Biobrick would not be induced until tetrathionate was detectable in the local environment. The Biobrick, to our knowledge, does not provide any additional ability to produce products with the capacity to regulate the immune system, aid its survival in the environment or persevere in special storage conditions such as within an aerosol.

In the long term it is unknown how the production of this Biobrick would affect the population of C. difficile. A kill switch is not being introduced into the Biobrick as it is hypothesised that once activated through inflammation, levels of C. difficile will be decreased to the point where inflammation ceases. It seems unlikely that every individual C. difficile cell will be wiped out but instead levels will be kept under the control of the returning normal gut flora levels. It may be possible that C. difficile could build up resistance to the endolysin through alteration of its cell wall binding domain recognised by the endolysin (Mayer et al. 2008). We also decided against a kill switch as most kill switches lyse the cells and so in the gut this could potentially result in DNA fragments being released into an environment containing bacterial cells naturally competent for DNA uptake. This could therefore have the potential consequence of spreading synthetic DNA into the natural environment, which should of course be avoided.
The laboratory in which this work was carried out is a controlled environment and requires secure gain of access, reducing the possibility for unauthorised persons to enter.

Environmental safety
It is well known that E. coli K12 strains are unable to sufficiently survive in the natural environment outside laboratory conditions (Na et al. 2006). They are unable to produce spores, harbour no ability to vaporize, cannot survive well in soil, seawater or air and so it would be unlikely for these organisms to proliferate in the outside environment. E. coli K12 strains are also themselves unable to colonise the gut, so if this project were to be developed further, it is unlikely that this would cause any problems ( EPA. 1997 ).
The Biobrick produces no known products that can play any significant role in the environment or environmental processes. The endolysin fused onto the VgrG protein is specific for C. difficile and should not pose any threat to plants or animals.
It may be possible that any number of the bioparts can change function through mutation, in which case the outcome would be unpredictable and any number of scenarios may occur. Due to the lack of homology with the proteins produced in this project and those of pathogenic organisms, the chances of gaining a harmful ability appears to be slim. The most likely outcome would be that a loss of function would arise.

Question 2 - Do any of the new Bio-Brick parts (or devices) that you made this year raise any safety issues?

No. Extra safety issues for each Biobrick part were unnecessary and therefore not included upon submitting them to the Registry.

Some Type VI Secretion Systems are thought to have activity against eukaryotic cells. The Type VI Secretion System that we have engineered originates from Salmonella LT2 and is located in pathogenicity island-6 (SPI-6). It is regarded that this protein secretion system is not involved in virulence against animal hosts, but normally has an anti-bacterial activity (Blondel et al. 2009). In any case, our engineered construct contains the structural genes for the secretion system only and does not encode any secreted effectors or other virulence factors (Blondel et al. 2009).

Question 3 - Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?

Yes, the University of Dundee requires comprehensive risk assessments to be carried out prior to the start of any project. Any accidents or spillages of live micro-organisms must be reported and correct disposal of waste in accordance with University regulations must be ensured at all times.

The details of this project were discussed with both the Health & Safety Coordinator and the Information Officer for the College of Life Sciences and a risk assessment specific to our project was then provided ( 2012 risk assessment ). This GMO risk assessment was consequently approved by the College of Life Sciences Biological Safety Committee. The overall project is deemed as class 2 overall due to modification of S. typhimurium for the western blot analysis experiments. The college possesses a generic notification for HSE (Health and Safety Executive) that covers this project. The safe operating procedures for level 1 ( sop 1, 2012 ) using E. coliand level 2 ( sop 2, 2012 ) ,working with S. typhimurium were also provided and adhered to throughout the project.

Before commencing our outreach day at The Shore, discussions were held with the Health and Safety Coordinator and also Dr Nicola Stanley-Wall, a PI in the Division with valued experience of holding public events. A full risk assessment was carried out in conjunction with them to ensure all safety standards were met outside of the laboratory ( agar risk assessment 2011 ).

The following guideline procedures apply in Scotland:
Genetic Modification Legislation from the Scottish Government ( Scottish Government 2011 ).
Biosafety guidance from the Health & Safety Executive ( HSE ).

Question 4 - Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

It is important to stress that safety is extremely important for those working in a laboratory environment and needs to be a high priority from the very start. Security should play a big role in iGEM. Engineered organisms must be safely out of reach from members of the public, thus preventing the risk of contamination with the outside environment.

Regular worksheets could be filled in by a member of the team who assumes overall safety of the project and submits any accidents or spillages. This ensures that all safety regulations are being carried out by all members of the team. It may also be an idea to submit photos/videos of work stations when asked by iGEM providing physical evidence of the working environment of each team.

When biobricks are uploaded onto the registry the system appears to accept anything you put. It remains unclear as to whether any checks occur or if the system just takes your word for it. It should be made clear that the uploaded information will be scrutinised before the information is posted live for all to see.