1) Safety Level Rating System

In Germany, the classification of the biological safety level is regulated by the genetic engineering law (Gentechnikgesetz, GenTG) and is elaborated by the genetic engineering safety regulation law (Gentechnik-Sicherheitsverordnung, GenTSV). In detail, these laws set the juristic safety-standards to the studies of genetically modified organisms (Gentechnisch veränderte Organismen, GVO) and the locations where these studies are carried out.

There are four levels of security, with the fourth being the one with highest security standards. Each level is based on the lower ones, so that, for example, the rules of the first, second and third level also apply to the fourth level.

The laboratories are named after the safety standards that are required to work in them: S1-S4. We only operated on the first safety level, thus we worked in S1-Laboratories under S1 circumstances.

The S1-safety level is characterized by working with organisms, that, when treated with proper care and when operating with the appropriate safety measures, can not pose a threat to the environment and healthy people.
The organisms that we worked with can be classified according to that.

With the friendly allowance of the Institute of Synthetic Biology (Prof. Dr. Matias Zurbriggen), the Institute of Microbiology (Prof. Dr. Michael Feldbrügge), the Institute of Plant Biochemistry (Prof. Dr. Andreas Weber) and the Institute of Biochemistry I (Prof. Dr. Lutz Schmitt, PD Dr. Ulrich Schulte) at the HHU we were able to push forward our research in a safe and successful way. In the institutes, we always received help by their members if we needed it, they made our work much more efficient and enjoyable.

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2) Work Environment

In all laboratories, there are certain restraints: One may not eat, drink, smoke, put on make up or store food inside the laboratories. Furthermore, one is not allowed to work completely alone, pipet with one’s mouth or operate with open wounds or bandages. We stuck to these rules strictly.

In our laboratories, there were workplaces especially for writing (protocolling), that are separated from the surfaces where work with GMO’s is carried out. Sinks with running cold and warm water were present. The surfaces that were used for working were easy to clean.
While working in the laboratory, windows were closed.
We avoided the formation of aerosol all the time.
Before and after working with clean benches, centrifuges and other equipment, we disinfected them thoroughly.
All institutes we worked in were equipped with autoclaves for disinfecting all kinds of waste and working equipment like pipette tips and microtubes.

Particularly dangerous chemicals like ethidium bromide (EtBr) for gel electrophoresis are only used in specialized rooms to protect everyone that does not work in them.

Most of our work was carried out on open work benches while complying with the regulations.

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3) Personal Protective Equipment

While working in the laboratory, we wore lab coats with long sleeves that are made of 100% cotton, closed shoes, safety goggles that cover the eyes frontal and sideways at all times to protect ourselves.

We wore nitrile gloves when working with substances that required a self-protection and tied up our hair when they were too long while working in the laboratory.

While working with EtBr, we used thicker nitrile gloves to protect ourselves from this dangerous chemical.

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4) Disposal of Biological Waste

Also sticking to the law for working in S2 laboratories, we disposed waste, that has been contaminated with genetically modified organisms in specialized plastic bags that can be sterilized with an autoclave to make it harmless. After this process, the waste could be disposed safely.
Liquid waste was collected in special containers that were autoclaved as well.

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5) Personal safety training

We received a safety training for each laboratory we worked in by either safety advisors or experienced members of the institute.

• Institute of Microbiology: Ute Gengenbacher
• Institute of Plant Chemistry: Samantha Kurz
• Institute of Synthetic Biology: Prof. Dr. Matias Zurbriggen
• Institute of Biochemistry I: PD Dr. Ulrich Schulte

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6) Organisms and parts that were used

Organisms:

Species name	Risk Group	Risk Group Source	Disease risk to humans?
E.coli K12	1	Source	No
S.cerevisiae INV Sc1	1	Source	No
Hela cells	1	Source	No
HEK2P3T	1	Source	No
CHO Cells	1	Source	No

Parts:

Part number/name	Natural function of part	How did you acquire it?	How will you use it?
Pdz-mcherry-bax184	Apoptosis through Bax protein	Lab	Testing the optogenetic parts in vivo
Lov2-gfp-tom5	Locating proteins to mitochondria	IDT	Testing the optogenetic parts in vivo
Pdz-mcherry	Red light	Lab	Testing the optogenetic parts in vivo
tdh3-rbs-lov2-gfp-tom5-hh01-araC-rbs-pdz-mcherry	Locating proteins to mitochondria and apoptosis through an anchor protein	IDT, Biobrick	Testing the optogenetic parts in vivo
tdh3-rbs-lov2-gfp-tom5-hh01	locating proteins to mitochondria	IDT, Biobrick	Testing the optogenetic parts in vivo
araC-rbs-pdz-mcherry-bax184e-hh01	Apoptosis through Bax protein, inducible through araC prmotor	Biobrick, Lab	Testing the optogenetic parts in vivo
TetO-tetR-PIF6-Phybr-VP16	Complete transcription control-system	IDT, Lab	Testing the optogenetic parts in vivo
tdh3-rbs-lov2-gfp-tom5-hh01-araC-rbs-pdz-mcherry	Locating proteins to mitochondria and apoptosis through an anchor protein	IDT, Biobrick	Testing the optogenetic parts in vivo
TetO-tetR-PIF6-Phybr-VP16	Complete transcription control-system	IDT, Lab	Testing the optogenetic parts in vivo
tdh3-rbs-lov2-gfp-tom5-hh01-araC-rbs-pdz-mcherry	Locating proteins to mitochondria and apoptosis through an anchor protein	IDT	Testing the optogenetic parts in vivo
TetO-tetR-PIF6-Phybr-VP16	Complete transcription control-system	IDT	Testing the optogenetic parts in vivo
tdh3-rbs-lov2-gfp-tom5-hh01-araC-rbs-pdz-mcherry	Locating proteins to mitochondria and apoptosis through an anchor protein	IDT	Testing the optogenetic parts in vivo

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7) Risks of our Project

Risk to the Safety and Health of Team Members/People Working in the Lab

E. coli has a low, but not non-existent virulence. For this reason, we wear gloves, lab coats and safety goggles while operating in the lab. Moreover, we wash our hands when entering and leaving the lab to eliminate every risk of carrying the organisms out of the lab.

Also, we disinfected the clean bench every time before and after the use to minimize the risk for the people who want to operate on it afterwards.

Nevertheless, a little risk remains when operating with inflectional bacteria or dangerous chemicals like EtBr, which can manipulate the genome on the surface it reaches.

We did everything we could to eliminate all risks.

Risk to the safety and health of the General Public

To protect the people outside the laboratory from the dangers that we deal with, we wore safety equipment and stuck to all the rules and regulations of S1-Laboratories so that they do not come in contact with dangerous chemicals or genetically modified organisms.

An exceptionally important aspect of the risk to public health is that we operated with antibiotics. When exposed to excessive amounts of antibiotics, microorganisms may develop resistances against them, making it more difficult to treat diseases caused by them. For this reason, it was of important concern to ensure that no antibiotics enter the water supply of the public by getting into the drain.

We always made sure to make them harmless and minimizing the risk by putting it into the autoclave.

Risk to the environment

As mentioned above, we treated every dangerous chemical and modified organisms with special care and disposed them appropriately, so they can not be released into the environment.

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8) Risks of our Project in the future

The main reason why we are working on this project is to help people suffering from cancer. Developing a drug that works with the help of a light switch would take a long time before it could finally be used to treat cancer and even though it has to undergo different preclinical and clinical studies, it is not guaranteed that no side effects occur during the treatment. Besides targeting cancer cells, also healthy tissues could be targeted by the drug.

The goal is to increase the specificity of the drug to cancer cells, but in the end it cannot be ensured that no healthy cell will be affected. Therefore there is a risk that, if someone, who has bad intentions, has access to the drug, it could be used for harmful purposes even though the drug should be specific enough for targeting only cancer cells.

If the drug does not only affect cancer cells but also healthy cells it can damage tissues and cause side effects and also other symptoms of illnesses can arise. People with bad intentions could modify the drug so that it targets preferentially healthy cells and therefore harm people, but that would also need blue and red light in a sufficient intensity that would activate the “kill switch”. But only the possibility that it could be used for harmful purposes raises ethical concerns in people.

Can the drug really reach a specificity for mainly targeting cancer cells so that the side effects can be kept on such a low level that it is worth it to use this drug for cancer treatment? Can it be ensured that the drug does not fall into wrong hands? If it does not work, how about all the preclinical and clinical studies, where the drug was tested on animals and humans who suffered from side effects to determine the drug’s efficiency and effects?

Animal testing is always a topic which raises ethical concern in people. Many experiments cause pain to the animals or reduce their quality of life in other ways. Many people agree, that if there would exist alternative testing methods that reveal equally valid results animal testing should stop. But until this kind of experiments are developed experiments on animals should be made as human as possible. ^[1]

Unfortunately animal testing is necessary before the drug can be tested on humans. Testing the drug on cell cultures does not reveal the needed results. It has to be ensured that despite the enormous complexity of a living body the amount of off- target effects is as low as possible. But even if the animal testing reveals good results, it is still not guaranteed that the drug works the way it should in human.

Furthermore the drug could raise criticism because it is based on a so called “kill switch”. People who do not know exactly what this mean could refuse a treatment even though it would be a useful treatment. Establishing a new drug always takes time because the method has to be accepted by the patients and the patients have to be willing to be treated with this drug. And yet it cannot be guaranteed that this therapy would have less side effects than the usual and already developed ways of cancer treatment.

To use our system to harm people would be very unadvantageous as our aim is to reduce off- target effects and utilize the so called “kill- switch” to increase safety and targeting due to the fact that the targeted cells have to shedded by the specific wavelengths. Furthermore it takes an extra effort to get the light to the wanted cells.

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References

^[1]= https://www.vfa.de/de/arzneimittel-forschung/so-funktioniert-pharmaforschung/so-entsteht-ein-medikament.html