Safety
Read more about how safety was integrated in all lab work.
Biosafety
Biosafety Training
All of the team members that are part of the Aalto-Helsinki team passed two theoretical and one in person laboratory safety courses, prior to being allowed to enter the building from the School of chemical Engineering in Aalto University. This is part of the regulations in Aalto University and required of anyone working in the building of the School of chemical Engineering to ensure proper and safe working. The training covered the following topics:
- Emergency procedures
- Recycling waste and disposal of biological waste and lab consumables
- Chemical, fire and electrical safety
- Difference between biosafety levels
- Biosafety equipment
- Lab rules and equipment for personal protection (clothing, eating, drinking, etc.)
- Responsible individuals (i.e. lab technicians and access to the list of lab equipment instructors)
- Disinfection and Sterilisation
For any other laboratory device that were utilised by the team in the lab an additional safety instruction was given prior to using the device ensuring the proper usage of the device as well as personal protection from the device. These devices included:
- Lyophilizer
- SDS-PAGE
- Sonicator
Biosafety Level
The lab our group is working in is a GMO Biosafety Level 1 lab.
Work Space
Our workspace consists of three fume hoods, two laminar flows and nine benches. We ensure to not work with ethidium bromide in our lab space, but use a different lab dedicated to that work. Furthermore, other basic equipment is available to us like centrifuges, PCR machines, incubators, heat blocks and more. However, for specialised equipment like the lyophilizer or sonicator we will access other lab spaces in the facility of the School of chemical Engineering building of Aalto University. For the duration of our work we have been able to store the BioXp by Codex in our lab space. Additional work spaces included Ville Paavilainen's lab in which we started the first round of ribosome display to ensure a first supervised experiment. We only worked on one provided bench in this lab, which was equipped with all necessary safety measurements.
Personal Protective Equipment
By the regulations of Aalto University; anyone working in the lab has to wear personal protective clothes including a lab coat, goggles and gloves. The gloves can differ according to the work performed and the safety concern caused. However, since we work on a low safety concern project when in regards to heat, cold or other agents, we wear nitrile gloves for all work performed.
Chassis
The strains worked with included the approved BSL1 strains TOP10 E. coli and 5-alpha E. coli.
Specific Risks
Our project does not include any specific risks like animal use, gene drive, nor human experimentation and we only work with BSL1 organisms present on the white list. The main safety related risk that we have been targeting is related to the inter-country transfer of our synthetically produced DARPin. We expressed, purified and dry-freezed a GFP-targeting DARPin that was transported by us via a commercial passenger flight to Münster, Germany from where the TU Dresden Team would transport it to their laboratory.
In order to assess that the transport from Finland to Germany via a commercial flight was feasible we contacted several departments involving customs in Dresden, REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) from the European Union regulation as well as the iGEM foundation. REACH advised us that there are no current regulations for the transport of a synthetic protein in the EU under the weight of 1 tonne. Since our protein was only a few µg we were able to transport it via Finnair to Germany. We had informed Finnair beforehand of our plan and received confirmation that we were allowed to transport the protein to Germany via a commercial flight in their company
The dangers of our DARPins are that they were randomly designed and we do not know their specific target; therefore, they might be able to bind to different targets that we are unaware of. However, the DARPin that we gave the TU Dresden team was a GFP-targeting DARPin, which would not have posed a risk during the transport. For the other DARPins, a potential risk may occur for an unidentified binding target. However, these DARPins will never be released beyond containment and only worked within the lab.
Additionally, we are also creating a GMO (genetically modified organism) for our bioreporter in E. coli. However, we informed ourselves of all safety regulations regarding GMOs in Finland listed below. GMOs pose a risk when released beyond containment. However, our GMO would also never leave the lab even in a real-life application, because it would only be used to measure quorum sensing in patient's or biofilm samples in the lab.
A risk of our project concerns the effect of our product. Biofilms are a very novel topic and the term has only been coined in 1978 (Costerton et al., 1978). Therefore, there is some discrepancy in the research and there are also hypotheses stating the opposite that induced quorum sensing contributes to the shedding, while no quorum sensing leads to build-up of the biofilm (Boles and Horswill, 2008). Our product may be affected by this, but all articles state that downstream activity is lowered when the quorum sensing system is inhibited (Le and Otto, 2015). This is an important statement, because the downstream genes encode for virulence factors (Kong et al 2006). Therefore, decreased quorum sensing also involves decreased virulence. Biofilms in itself can be advantageous, because they reduce the virulence of bacteria. Therefore, our DARPin termed QBlock is giving an ideal advantage in treatment when preventing the wound from further infecting the wound.
Data Safety
We contacted and interviewed several clinical staff members at the Helsinki University Hospital Area (HUS) Wound Centre. These included regenerative medicine professor and researcher Dr. Esko Kankuri, dermatologist Dr. Heli Lagus, plastic surgeon Dr. Mila Kallio as well as two wound care nurses. The information they disclosed to us about general patient experiences, diagnostics, treatment options and challenges of wound healing were recorded in our notebook and our Google drive only and not shared beyond our team, with agreed uses of the information being to (i) inform our project's direction further (ii) write about human practices and the chronic wound burden in our blog, poster and presentation material. No patients were named in any recorded material. Additionally to clinical staff, our team wanted to interview chronic wound patients to understand the problem better. We contacted the HUS ethical committee in the hope that we could visit the hospital for the interviews. For the ethical committee, we prepared a consent form where we stated the purpose of the patient interviews and a list of possible questions. However we did not receive permission to record any interviews or interview the patients at the hospital. Instead, we were allowed to have the survey if the doctors asked the questions or the patients themselves contacted us.
Regulation of GMOs in Finland
The Gene Technology Act (377/1995) regulates the use of GMOs in Finland, and we have been basing our biosafety precautions on this legislation. All legislation mentioned, including the Gene Technology Act, can be found here.
The aim of the Act is to promote the safe use and development of gene technology in accordance with the precautionary principle and in an ethically acceptable way, and to protect human and animal health and the environment when contained use or deliberate release into the environment of GMOs is carried out.
The Gene Technology Act is based on the EU Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms and on the Directive 2009/41/EC on the contained use of genetically modified micro-organisms.
The following definitions are applicable to our project in its current developing form as well as its prospects for future scale-up:
- micro-organism means any microbiological entity, cellular or non-cellular, capable of replication or of transferring genetic material, including viruses, viroids, animal and plant cells in culture and human cells and human tissues in culture;
- genetically modified micro-organism means a micro-organism in which the genetic material has been altered in a way that does not occur naturally by mating or natural recombination;
- risk assessment means evaluation of the risks that the use of genetically modified organisms may constitute to human and animal health and the environment, whether they are direct or indirect or immediate or delayed; and
- product means a preparation placed on the market that consists of genetically modified organisms or a combination of them or that contains them.
Under Chapter 8 the Gene Technology Act, Contained Use of Genetically Modified Organisms (10.9.2004/847), our project remains under risk class 1 or 2. This Chapter is relevant to us as it details the classes of use and appropriate containment levels guided by the adequate protection of human and animal health and the environment. The four classes are as follows:
- activities under class 1 are linked with no or negligible risk, for which level 1 containment is sufficient;
- activities under class 2 are linked with a low risk, for which level 2 containment is sufficient;
- activities under class 3 are linked with a moderate risk, for which level 3 containment is sufficient; and
- activities under class 4 are linked with a high risk, for which level 4 containment is required.
The Gene Technology Act is supplemented by Government Decrees and Decrees of the Ministry of Social Affairs and Health. The following are relevant for us:
No. 1053/2005
Decree of the Ministry of Social Affairs and Health on principles of risk assessment of the contained use of genetically modified micro-organisms, on classification of the contained use, and on containment and other protective measures
- Section 2: General principles of risk assessment
- Section 3: The stages of risk assessment
- Section 4: Identification of harmful properties
- Section 9: Culture conditions in evaluation of the likelihood of harmful effects
Biosecurity
Synthetic Biology
Our project consists of production of two different synthetic biology products: a DARPin and a bioreporter. We carefully chose methods and target sites to minimise the risks associated with these products.
DARPins
Our project's main idea is to produce DARPins to prevent further biofilm build-up in wounds. DARPins are small synthetic proteins and hence have the same possible problems as other proteins. Aggregation and off-target binding are such problems that will be elaborated in below (Xie, Xie and Bourne, 2011 & Dimitrov, 2012). We acknowledge the possible biosecurity hazards that could be caused by our protein and how we are preventing them from happening. We wanted to design our product so that it would have no risks of misuse and unwanted effects. Biosecurity views were considered during the whole process starting from ideation process in Spring and ending with storage of our produced DARPins at the end of our project.
Replication:
Our DARPins are proteins, therefore they cannot replicate by themselves. We decided to work with proteins for this
reason.
Aggregation:
We are aware that some proteins can have aggregation prone properties (Dimitrov, 2012). The possible aggregation process
would be possible to study when there are further testing opportunities available. At the same time the ideal
environment for the protein would be studied and then decided if our protein would be suitable to work in human skin.
Another option would be in silico approaches such as an algorithm that would predict the possible aggregation.
Unfortunately, our team did not have time to run our DARPin sequence through that kind of algorithm (Navarro & Ventura,
2022). However, we plan to use computational methods in product development.
Binding:
Another possible problem is that our DARPin binds to an unwanted binding site such as a receptor in an epithelial cell.
For further studies, we would like to test our DARPin with computational methods in silico. And in later stages in
animal models and clinical trials. In addition, the efficiency of the binding could be tested by comparing our DARPins
against different databases in order to find out if they are similar to any human protein that has known binding
functions in the human body. In that case, we would not want to proceed with that protein to prevent any unwanted
binding.
DNA
To produce our DARPins we created a DARPin library that consists of our DARPins' DNA sequences. The possible problems are both the misuse of the DARPin library method to produce peptides that do have harmful effects and the misuse of our bioreporter.
Gene Drive
Our DARPin targets the AIP1 of S.epidermidis. In this way, we aim to prevent the formation of further biofilm instead of aiming to kill the bacteria completely. We are aware that killing the bacteria could lead to a faster rate of evolution of the bacteria to become resistant towards our treatment (Munita & Arias, 2016).
References
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Agr-mediated dispersal of Staphylococcus aureus biofilmsPLoS pathogens, 4(4), p.e1000052
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How bacteria stick.Scientific American 238, no. 1: 86-95
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Therapeutic proteinsMethods Mol Biol. 899:1-26
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Staphylococcus quorum sensing in biofilm formation and infectionInternational journal of medical microbiology, 296(2-3), pp.133-139
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Quorum-sensing regulation in staphylococci—an overviewFrontiers in microbiology, 6, p.1174
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Computational methods to predict protein aggregationCurrent Opinion in Structural Biology. Vol. 73
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Mechanisms of Antibiotic ResistanceMicrobiol Spectr.(2):10.1128
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Structure-based systems biology for analyzing off-target bindingCurr Opin Struct Biol. 21(2):189-99