Team:Newcastle/Notebook/Safety

Safety

Have we met the iGEM safety requirements?

We checked our organisms against the white list to see if we needed to complete a check-in. We found that we did not as our two organisms E. coli K-12, and S. cerevisiae were on the white list.
We checked the components of our parts against the white list to see if we needed to complete a check in. We found that we did not as we use only non-red flagged registry parts or coding sequences from the list of dangerous categories.
Completed the ‘About Our Lab’ questionnaire.
Completed the ‘About Our Project’ questionnaire.
Completed the final safety form.

Our Local Guidelines

As a UK based team we familiarised ourselves with the regulations in the UK concerning the proper safe management of biological organisms, and particularly GMOs. In particular we found that all work involving biological material in the UK is subject to the Control of Substances Hazardous to Health Regulations 2002(COSHH). The specific schedule relating to our work is Schedule 3, ‘Additional Provisions Relating To Work With Biological Agents’. Additionally, because we are working with genetically modified organisms (GMOs) our work is subject to The Genetically Modified Organisms (Contained Use) Regulations 2014 which is the legislation implementing the European directive on on the contained use of genetically modified micro-organisms. This legislation is summarised in documents by the UK Health & Safety Executive.

In essence we determined that the aim of these regulations is to:

identify the hazards and assessing the risks to health arising from the use of hazardous substances during a work activity.
decide what control measures are required to prevent or control exposure
ensure that control measures are properly implemented and maintained
monitor the exposure of users to hazardous substances if appropriate
providing information, instruction and training to users of hazardous substances to ensure safe working practices and competency
ensuring sufficient arrangements to deal with any accidents, incidents and emergencies that are reasonably foreseeable

We also familiarised ourselves with our Institution's biological risk assessment policy and chemical risk assessment policy to ensure we completed our risk assessments correctly when required.

Safe Design

It is often said in Synthetic Biology that the emergent behaviour of genetic systems are often not immediately obvious. Consequently, responsible design is an important part of synthetic biology, so as part of our design work we decided upon a set of criteria which we would use to assess each design for risk. We settled on the following list of criteria:

The first set of questions should be answered negatively for any of our designs to ensure that we meet our safety aims and those of iGEM.

Do any of the coding sequences for proteins in the design come from organisms listed as hazardous or are found in Risk Group 3 or 4?
Are any of the coding sequences from virulence factors, or would they mimic the behaviour of an existing virulence factor?

We then examine if the design might pose any additional risks by considering the following questions. If the response to any of these is yes, then a further assessment of the risks posed by the design needs to be performed.

Is the chassis organism from a risk group other than Class 1 (bacteria) or may it be harmful (non bacterial chassis)?
Are any of the coding sequences from risk group 2 organisms?
Would the system enable the dissemination of genetic material?
Can a mutation result in a dangerous organism?
Does our device present any novel risks?
Are any of the coding sequences used sourced from places other than the iGEM registry?

We answered these for all of our constructs and found the only construct that needed further risk assessment was our controlled OrpF expression device (BBa_K1895005). This is because that part contains a coding sequence, OrpF, from a risk group 2 organism (Pseudomonas fluorescens). To assess this risk we identified a number of ‘mitigating factors’ that would minimise the risk posed by using this sequence.

The protein we are dealing with is well characterised.
The protein does not play a role in toxicity or virulence.
The protein will only be produced at high levels in growth media containing L-arabinose.

Based on these, we believe that the risk from using this sequence is negligible and so continued with this design. Had we not identified any mitigating factors we would have sought to use an alternative porin from a safer organism. We discuss the further steps we took to minimise the risk when working with this organism in the section on safe lab work.

The other main risk we identified for our project is physical containment failure. In order to assess this risk, we need to know both the consequence and likelihood of the risk occurring. It can then be assigned a risk category using a risk assessment matrix.

Risk Matrix Diagram — Figure 1: The risk matrix we use to assess the risks in our project.

The Paris Bettencourt 2012 iGEM team have shown how the likelihood of a physical containment failure can be estimated. If our physical containment fails then E. coli will be able to enter the environment, from a risk perspective this is a ‘failure’ regardless of the rate or amount of bacteria that leak. Therefore for us, the likelihood of a physical failure is related to the chance of our microfluidics chambers leaking. In our experiments measuring the temperature effects in the chamber we found that 1 in 10 chambers failed in use. This is a high chance of failure which we are working to mitigate using alternative materials in the construction of our chambers (plastic rather than PDMS).

The effect of a physical containment failure is low, the volumes of E. coli (0.1ml) involved are easily contained in the laboratory (which is where we intend our devices to be used) and the cells can be lysed with 70% ethanol. This results in an overall risk of ‘medium/low’ at this time. However, in the future, we hope that our system might be taken out of the lab. For this reason, we further investigated ways in which we could improve the safety of our system.

In investigating how we could further increase the safety of our transformed organisms we came across the use of genetic kill switches which have been investigated by a number of iGEM teams in the past, e.g. Wageningen 2014 , the parts for which can be found in the parts registry. We considered adding such a system to our own bacteria but felt that this increased the complexity of our system too much. Additionally, in cases such as the microbial fuel cell, we are explicitly trying to reduce the metabolic strain on the cell which would not be helped by adding a kill switch system.

After further consideration of this issue we identified an alternative safety system which functions along similar principles to a ‘kill switch’ and which could easily be integrated into our systems to make the safer. That is, the notation of ‘dependent bacteria’. These are strains which require supplements without which they could not survive. An example of such a strain mutation is ΔdapD. Strains with this mutation in the gene encoding Succinyl diaminopimelate aminotransferase require a succinate supplement in order to survive. Refer to the OpenWetWare page on E. coli strains for more information.

Such a dependency system is summarised in 'Building-in biosafety for synthetic biology'; (Wright, Stan & Ellis, 2013) and does not appear to have any negative impacts on the bacteria in our use case. As we do not have time to test such as system as part of our own work we located an existing experiment in the literature to serve as a proof of concept for this scheme. The work by Cranenburgh (2001), ‘Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration’ shows that ΔdapD mutants suffer low growth without supplements (in this case IPTG as dapD was placed under the control of the lac promoter). We have included the relevant figure below (Figure 2), the line with squares in graph B charts the growth of the mutants with no supplements.

DAPD Mutant Growth Graph — Figure 2: Graph showing growth rate in *ΔdapD* mutants. The line with squares in graph B shows that there is reduced growth of the mutants when no supplements are present.

In the future we suggest that our constructs be used in bacteria with this or a similar mutation to improve the safety of our system. This serves to supplement our physical containment strategy.

Whilst considering the risks posed by our physical container, it occurred to us that we needed to also consider more general impact of creating a physical product such as our plug ‘n’ play kit on the environment and users. Because our system is intended for lab use, at the end of its life it will be sterilised and then discarded. Our PDMS chambers can not be recycled, and so would have to be sent to landfill. As PDMS degrades in soil, it's non-toxic and does not bioaccumulate in sediment dwelling organisms, this is an acceptable disposal method. The remaining components of our kit are ABS plastic or glass and are recyclable provided the PDMS is removed. We will supply information on how to remove the PDMS and this recycling information alongside our kit.

Safe Lab Work

As part of our laboratory induction which took place during the first week of our project we were introduced to the General Safety Procedures for working in our labs. There was a focus on microbiological safety, and, as we would be working in both class 1 and class 2 biological safety level labs, the differences for working in each. We covered biological safety cabinet use and aspectic working techniques, the use of protective clothing and Personal Protection Equipment (PPE), disinfection and waste disposal.

In addition to biological safety we also covered chemical safety, including how to contain a chemical spill and the requirements for bringing new chemicals into the laboratory. Finally, we also were introduced to working safely with electrical and ultraviolet equipment.

During our work we required chemicals that had not been used in our lab before. For all of these we located the required material safety data sheets which are collected below.

For those chemicals which we deemed to be hazardous to health, like Methylene Blue we completed the appropriate University risk assessment form using the information from these safety data sheets. As well as considering the following factors:

The level for a harmful dose.
Stability of the material.
The chemical's ability to spread and ease of containment.
The concentration and volume we would be using.
How readily available treatment is in the event of an accident (e.g. eyewash stations, etc.).

From a biological safety perspective the most important risk management practices depend on the risk group of the organism you are working with. We were working with Risk Group 1 Organisms i.e. ‘those which are not associated with disease in healthy adult humans’. This means the main techniques we used to manage our risk when using these was wearing the appropriate PPE (such as nitrile gloves and lab coats) and proper disinfection of surfaces and equipment before and after use with ChemGene and 70% ethanol during our project.

As our work involves genetic modification, we end up with ‘new’ organisms and so we are responsible for assigning them a risk group. This risk group is assigned depending on the components that were used in making the new organism. The organism, protein and risk group for parts not from E. coli are given in the table below.

Bacteria	Protein	Risk Group
Pseudomonas fluorescens	Outer membrane porin F.	2
Synechococcus elongatus	Metallothionein SmtA.	1

As one of the organisms we use is a risk group 2 organism, it was important that we closely reviewed the coding sequence we intended to use. In this case, it is OprF from Pseudomonas fluorescens. Fortunately, when we consulted the UniProt entry for OprF (ID: PORF_PSEAE) we found that this protein is well characterised, with experimental evidence at the protein level. Additionally, the structure of some of its domains has been determined using crystallography and can be found in the protein data bank.

According to the available data we know that OprF has porin activity and serves to form small water-filled channels which are involved in ion transport. The protein also has a structural role in regulating cell shape and the cell's ability to grow in low-osmolarity medium.

We assess this as being a safe to use protein coding sequence as it does not fall into one of the dangerous categories listed in the safety guidelines, nor when we consulted the database of virulence factors for Pseudomonas did we find the gene for this protein indicated. Additionally, the gene product, the porin itself has no potential for dual use and is not harmful to the environment in any way.

Although we assessed this coding sequence to be safe for use in our designs, we have chosen to assign risk group 2 status to E. coli transformed with plasmids containing constructs which contain this gene and express the OprF protein. That is, our part BBa_K1895005. Any work involving this plasmid and transformed products will be confined to our level 2 biosafety laboratory.

Safe Shipment

None of the proteins in our constructs can be found on the Australia group list for export control or the US list of select agents so we do not anticipate any issues shipping our freeze-dried plasmid DNA to iGEM HQ. In keeping with best practices, we will be recording and tracking all the parts we ship.