1. Anticipation

The laboratory work for our project is carried out in ML-1 area lab spaces in the Bionanoscience department of the Faculty of Applied Sciences building on the campus of Delft University of Technology. An ML-1 space is equivalent to a BSL-1 space, which is considered the lowest level of microbiology laboratory biosecurity. This entails working with non-pathogenic microorganisms. All of our experimental work is conducted in ML-1 spaces, and mainly consists of molecular cloning, protein expression, protein purification and in vitro assays. Not all of the equipment we required was present in our own lab space, but was available to us in other ML-1/BSL-1 spaces of the department of Bionanoscience. To reassure knowledge for emergency operation procedures, it was important to have all of our team members pass a set of safety tests before starting any laboratory work. These tests included:

general building safety (meeting points, emergency numbers, escape routes, etc.)
general laboratory safety (chemicals, waste disposal, clothing, safety precautions, etc.)
biological safety (ML-1 grade safety, biological waste, safety precautions, etc.)
laser safety (general safety precautions)

Apart from these evaluated online tests, we also got instructions in person on how to work safely in the Department of Bionanoscience, where to discard what type of waste and how to minimize contamination risks not only within but also outside of the lab. For GMO regulations, we made sure our strains are maintained within our ML-1/BSL-1 laboratory space. This entails certain basic laboratory guidelines:

At any given time, individuals should wear minimal protective clothes (closed shoes, long sleeve shirts, long trouser legs, white lab coat). Depending on the experimental conditions, additional measures like gloves or protective eyewear might be needed;
When entering and leaving the BSL-1 laboratory space, individuals should wash their hands to prevent any unwanted spreading of contained organisms.
The working space is kept organized, tidy and clean;
Eating, drinking, smoking, presence/storage of nutritious material for consumption, application of cosmetics or contact lenses is prohibited.
Pipetting with your mouth is prohibited;
Presence of vermin is strictly prohibited;
Jackets, coats, bags, sweaters etc. (and other likewise personal belongings) should be stored outside the working space;
In case of contamination of surfaces, these should be cleaned and desinfected instantly;
Contaminated clothing should be directly autoclaved in case of contamination with biological agents/ GMOs through spilling.
Contaminated waste should be directed to designated areas for appropriate treatment thereof.

2. Safety of ADOPE

Next to general safety tests and instructions, anticipating views gained by prospective risk assessments and experimental planning contribute to a safe workspace. Therefore, our Safety Manager Kavish Kohabir wrote all required safety proposals for the experimental work to be done, with supervision and approval of Susanne Hage (Department Wetlab Coordinator) and Marinka Almering (Faculty Biological Safety Officer). Such proposals contain detailed information what biological material is used, how it is disposed of and registers whether these are conform ML-1/BSL-1 measures. An important additional element is a risk evaluation and a precautious view on possible incidents, injuries or other calamities. Furthermore, prospective planning allowed for insight on the legal borders of our project to make sure all of our work is conform the Dutch regulations and legislation concerning biosafety in the Netherlands. Needless to say, all of our work done also adheres to iGEM guidelines.

2.1 Biosafety: Work in ML-1/BSL-1 Laboratories

To minimize contamination risks and prevent as many incidents as possible, we consistently worked conform ML-1 rules in the department of Bionanoscience. Amongst others, this meant in ML-1 spaces there is: food/drinks may not enter the labs; no eating/drinking; certain clothing requirements (white laboratory coat, long trousers, no open shoes, hair tied); no storing of personal belongings like bags/jackets/sweaters; no plants/animals. Additionally, we cleaned the workspace with ethanol prior and after working to limit contamination risks. In special cases, like the in vitro work, we worked in special designated DNAse and RNAse free benches to prevent degradation of genetic materials used.

2.1.1 Hosts

Throughout the whole project, we demonstrate a recurrent motive to avoid any unnecessary risks. This is why, in the first place, we exclusively work with Escherichia coli as host organism for all the laboratory work. The used strains (DH5α, BL21 DE3 and BL21 AI) are considered non-pathogenic to humans (Risk Group 1) and thus are all on the so-called ‘iGEM White List’. The Safety Data Sheets provided by the manufacturers confirm that it is sufficiently safe to work under ML-1/BSL-1 circumstances with these organisms.

2.1.2 Vectors

For constructing strains and cloning genetic material into a strain, we only made use of plasmids as vectors. Most of the plasmids are derivatives of iGEM backbones, but we also cloned in pACYCDuet1™ derived plasmids for controlled expression of our constructs. Prior to cloning with this vector, we verified with the manufacturer’s Safety Data Sheet that it is safe enough to work with this under ML-1/BSL-1 circumstances.

2.1.3 Inserts

Prior to cloning, all the inserts (and combinations thereof) we wanted to use were all subjected to evaluation of potential safety risks. To stress the safety-by-design of our project, we made sure that there are no malicious combinations possible, thereby safeguarding the ML-1/BSL-1 grade of our project. For some of the biologicals, this required some additional efforts:

Our project makes use of the Tn5 transposase originating from Escherichia coli. Normally, a transposase is flanked by recognition sites called Mosaic Ends (MEs). This combination is called a transposon, which is a form of a mobile element. In our case, the Tn5 transposase is capable of recognizing these MEs, isolating the whole transposon and pasting it elsewhere. We made sure the coding sequence for the Tn5 transposase was not flanked by MEs at any time of the project. This was done to prevent uncoordinated migration of the transposon. For a similar reason, we verified absence of genomic MEs with the genome of the host strains, to prevent uncontrollable scrambling of a host’s genome. The only sequences that contained MEs in this project were linear fragments that were supplied to a host through transformation. Through this setup, we controlled as much as possible to make working with Tn5 transposase as safe as possible and sufficient to work under ML-1/BSL-1 laboratories.

Our project establishes a tool using CRISPR-Cas technology. We used a catalytically inactive variant of Cas9 called dxCas9. This means the machinery is incapable of inducing double strand breaks in a target sequence. Therefore, all of the strains created in this project are lacking gene drive possibilities.

We wanted to demonstrate the in vitro functionality of our construct on potential gene doping. As a case study, we focused on the human Erythropoietin gene EPO. The sequence we work with lacks all introns, as this is the scenario for gene doping DNA. The fusion construct is guided to gene doping DNA by gRNAs that target exon-exon junctions which are normally not present in native human DNA. Therefore, the gRNAs we use to target EPO from Homo sapiens would be regarded safe and harmless. As a confirmation, we received approval from the iGEM Safety and Security Committee to work with these gRNAs.

A demonstration of the functionality of our detection method would start from withdrawing gene-doped human blood, isolating DNA from it and then detecting it. However, hematological research on non-certified or unscreened blood can be dangerous and pathogenic if not handled with the corresponding ML-2/BSL-2 standards. Furthermore, even when using screened human blood (from e.g. a blood bank), we are bound to limitations regarding privacy. Due to the fact that our methodology makes use of sequencing, we could possibly unexpectedly detect background cell-free DNA sequences that hint for elevated epidemiological risks, or other valuable/sensitive data. In this case we would have to report this, regardless of the original donor’s interest. To prevent any of the above mentioned scenarios from happening, we chose to work with certified bovine serum (cow serum). We spiked this with gene doping DNA, extracted the DNA and performed our detection tests.

2.2 Safety Considering Work With Chemicals

Apart from working with biological materials, we also worked with a substantial amount of chemicals. Similarly, these chemicals were all evaluated prior to working with them, in order to estimate possible risks and work accordingly for safety measures. The Safety Data Sheets provided by manufacturers already contained a lot of crucial information for this. Examples of appropriate measures are: working with gloves in a confined designated area for SYBR Safe contaminated equipment; working with gloves and protective eyewear when handling gold nanoparticle generation in a chemical flow hood.

2.3 Disposal of Biologicals and Chemicals

A solid safety-by-design project already constrains a lot of possible risks, but is not a waterproof approach by itself. Appropriate disposal of materials used is a very important factor when it comes to containment of risks within the laboratory space. Therefore, all biological and chemical waste was treated accordingly:

2.3.1 Biological waste

Genetically modified organisms can alter natural ecosystem balances in a very unpredictable way. This is why containment of biologicals is one of the top priorities when keeping work in ML-1/BSL-1 spaces safe. All biological waste was collected in labelled bottles (ML-1 Waste) and sterilized by autoclaving. As a special exception, Bovine serum was collected in a separately labelled bottle (Animal Waste). It was the duty of our Safety Manager to maintain a balanced bookkeeping of incoming Animal materials and outgoing Animal Waste streams, and present this to the Biosafety Officer of the University.

2.3.2 Chemical waste

Exposure of hazardous chemical waste was done according to conventional guidelines in the department of Bionanoscience. This meant that we separated SYBR Safe waste, considered carcinogenic, in a separate tank. This was collected by a team of specialists in disposal of hazardous chemical laboratory waste. The same held for chemicals like Coomassie Blue, SYBR Safe or ethidium bromide stained agarose gels, but also for contaminated consumables.

2.4 Social Science Safety

Apart from laboratory work, we also conducted some social science research. Through distributing surveys, we managed to interview a over 250 people about their views on the interface between science and sports, and how extreme this may be. Since surveys are a way of experimenting with human subjects, we made sure that our way of operating complies all national and institutional rules. Hence, we did not ask for sensitive data other than age range, sex and level/direction of education. We have not requested any name, neither any other details for contacting the subject. The rest of the survey mainly consisted on closed or multiple choice questions on the opinion of the subject.

2.5 Safety by Design

Our project in essence already advocates for safety by the topic of gene doping detection. As a team, we thereby promote fair, healthy and especially safe sports practices. By investing in gene doping research, we aim to raise awareness of the seriousness of the phenomenon and possible negative futuristic consequences that we want to prevent.
The entire experimental planning done for laboratory work was conform the expression safety-by-design. As described above, we made great effort in avoiding all unnecessary safe risks by e.g. exchanging actual blood samples for certified bovine serum; controlling the abundance of Mosaic Ends to be recognized by the Tn5 transposase; controlled expression of novel constructs in non-pathogenic hosts.
When it comes to safety in our applied design, we have intensively been in contact with stakeholders from several relevant expertises. By sitting around the table with the National Institute for Public Health and the Environment(RIVM), for example, we realized having a cell-free application in the end would be the safest approach for our goal. Contact with the Dutch national doping authority informed us on the necessity of having a pre-screen in doping testing. This translated to a cell-free pre-screen approach, based on biochemical interactions of gold nanoparticles. The read-out is only based on color change and is easily accessible without complex laboratory equipment.

Team:TUDelft/HumanPractices/IntegratedHumanPractices