Difference between revisions of "Team:Heidelberg/Safety"

Safety and Security in General

---- am linken Rand https://static.igem.org/mediawiki/2017/5/5d/T--Heidelberg--2017_HP_Safety.png --------- In vivo directed evolution by PREDCEL and PACE is a powerful technology that requires to be handled with responsibility and care. Besides the very general safety precautions required for laboratory work with bacteriophages, one also needs to exclude potential risk of unwantedly evolving environmentally hazardous or even dangerous proteins.

SafetyNet- Preventing unintended Evolution of Hazardous Proteins

------------- rechts oben https://static.igem.org/mediawiki/2017/d/d6/T--Heidelberg--2017_HP_SafetyNet.png ------------- In vivo directed evolution by PREDCEL and PACE is a powerful technology that gives rise to nucleic acids and proteins with novel functions. The evolved functions thereby depend – in principle – only on the used gene sequence pool as well as the applied selection pressure. Therefore, before evolving sequences in our PACE and PREDCEL experiments, we wanted to confirm that no hazardous or dangerous products can unintentionally evolve. Therefore, each parental DNA sequence that was used in our evolution experiments was first investigated for hazardous/dangerous potential. To do this easily and efficiently, we created a software and corresponding web interface named SafetyNet, which scans any input sequence for similarity to pathogen-derived sequences, toxins or other sequences with the reported potential of being hazardous or dangerous.
We discussed our SafetyNet with Dr. Janausch and asked, which protein classes should be included in the list of hazardous sequences and he recommended us to test for oncogenic sequences too. Therefore we edited our SafetyNet to integrate oncogenes as potentially harmful sequences. We discussed the different safety aspects and Dr. Janausch concluded, that PACE and PREDCEL experiments should be performed in a risk group 1 laboratory using a high standard of good laboratory practice. Fortunately, our lab fulfills all safety and security aspects with regard to the experiments we perform during our iGEM project.

Protecting the Environment

---- rechts oben https://static.igem.org/mediawiki/2017/d/dc/T--Heidelberg--2017_HP_EcologicalandMedical.png ----- To ensure that we do not endanger the environment or ourselves we talked to Dr. Ingo Janausch, the representative for biological safety and security and gene technology at our university. Dr. Janausch made us aware of the fact that phages are volatile and therefore phage contamination can be a severe problem. Although phages are (like all viruses) challenging to work with, it is feasible to handle them safely provided good laboratory practice is applied with great awareness, Dr. Janausch stated. In our PACE or PREDCEL experiments, we exclusively work with phage genomes harboring a gene III knockout. Therefore, without the appropriate E. coli hosts, the phages cannot replicate and can hence be treated without high risk of unwanted phage spread. Surely, a functionalizing recombination between a gene III encoding plasmid present within an E. coli host cell and the recombinant phage genome could – in principle - result in replication competent wild-type phages. This event is, however, extremely unlikely and has not been reported yet in context of in vivo directed evolution as performed by us. The modified E. coli K12 strain, used for all PACE and PREDCEL experiments, is a nonpathogenic E. coli strain and therefore, biosafety is ensured when working with this strain. Furthermore, we carefully collected and autoclaved all (liquid) waste resulting from PACE and PREDCEL experiments and thereby ensured that the E. coli cells as well as the phages are destroyed after every completed experiment.

Protecting Ourselves

------ am rechten Rand https://static.igem.org/mediawiki/2017/e/e3/T--Heidelberg--2017_Safety-Phage.png ----- To meet the personal protection aspect, we were given the safety introduction for the facilities in our laboratory as well as the whole BioQuant building by Christiane Graffy the laboratory coordinator. Explanations of centrifuges, incubators, fume hoods as well as waste disposal of aqueous and organic liquids are only a few examples for the thorough introduction. Additional biosafety introduction for our S1 laboratory was conducted by Mareike Hoffmann one of our supervisors. Furthermore, we talked about dangers for the experimenter with href="https://2017.igem.org/Team:Heidelberg/Interviews">Dr. Janausch. First we noted that the phages and E. coli cells handled in PACE apparatus are “locked" in a tightly concealed system, thereby minimizing the risk of unwanted phage escape from the experimental setting. In contrast, in PREDCEL, the experimenter has to manually transfer the phage-containing supernatant from one flask to the next when starting a new PREDCEL cycle. We therefore decided to create special “PREDCEL" bench spaces to restrict the “open" handling of phages to specific areas. These areas were cleaned regularly and were equipped with separate waste containers, pipettes and (filter) tips. Also, we performed plague assays regularly from all equipment/substances handled within these areas to detect potential phage contaminations early on. As M13 phages do not represent a particular danger as compared to other biosafety level 1 organisms, personal protection with lab coats, glasses and gloves are sufficient for safe phage handling. However, href="https://2017.igem.org/Team:Heidelberg/Interviews">Dr. Janausch recommended to carefully consider the choice of laboratory gloves for experiments involving phages, as glove material with a large pore size could enable phages to quickly pass through. Therefore, we researched this issue and learned that tests for laboratory gloves used by us are usually performed with Phi-X 174 bacteriophage astmstandard. In our PACE and PREDCEL experiments we use the M13 bacteriophage that is 6.6 nm x 880 nm lee2012virus in size and therefore bigger compared to Phi-X174 bacteriophage with its 25 nm in diameter kazumori1981electron. Hence, we ensured that the gloves we use while handling phages represent efficient barriers for M13 phages used in PREDCEL and PACE. As side note: Avoiding any kind of unwanted phage contamination is absolutely crucial for the success of PACE and PREDCEL experiments. Any phages that can activate gene III expression on the attenuator plasmid present within an E. coli host cell during a PREDCEL or PACE runs can replicate and hence – in the worst case - outcompete the phage population intended to be evolved. Therefore, it is not only for safety reasons, but in the experimenter’s scientific interest to handle phages with great awareness and care.

Responsible Science

Legal Aspects of our Directed Evolution Approach
In order to approach our project from a legal point of view, we talked to Birgit Degner and Dr. Jens Degner, both judges within the ordinary jurisdiction of the German state of North Rhine-Westphalia with several years of experience. An in-depth assessment of the legal situation was not possible, because our project relates to several different fields of law such as environmental law, consumer protection, immission control, occupational safety, patent law and on a more general basis the Constitution, which in Germany is also called basic law. Nevertheless, they could give us a brief, personal opinion. We talked about in how far science, such as our project on directed evolution, currently is limited or enabled by German law. Birgit Degner and Dr. Jens Degner clarified that there are several legal limitations on what can be done in science, although art. 5, par. 3, line 1 of the German basic law generally ensures scientific freedom. This freedom though is limited by the other basic laws, such as in the first two sentences of the German Constitution: “Human dignity shall be inviolable. To respect and protect it shall be the duty of all state authority." (Art. 1, par. 1 of German Constitution). Based on this, several questions arise. In how far is directed evolution reconcilable with the human dignity? Is maybe the aspiration of individuality threatened by this new method? Is non-human life threatened and thus, must be protected by the scientist? In how far is the consumer able to choose between genetically modified products and non-modified products? All these questions have to be and have been considered by the lawmaker, leading to the creation of the German Act on Genetic Engineering in order to prevent and dissolve conflicts. As science progresses, e.g. with new scientific methods such as PACE and PREDCEL, the respective laws on science might have to be adjusted and reconsidered. With Germany being a member of the European Union (EU), this cannot just be done with national laws, but instead with EU laws. Birgit Degner and Dr. Jens Degner were not able to give us a confirmation that our project was fully in accordance with current German law, but they underlined that each member of our team needs to reflect the experiments done and assess whether they are in accordance with our own personal values. Apart from that, we need to ensure appropriate safety measures and precautions are taken in order to experiment safely. Whether we want to draft further principles for our project is a question we should answer for ourselves. They highlighted that science, especially in fundamental research like foundational advance, should, from their point of view, not be limited unnecessarily by law. Science lives from the human desire to gain a deeper knowledge and understanding of our world and this freedom for science should also be respected by law.

Ethical Concerns about Directed Evolution Approaches
Should humanity harness the process of evolution for their own benefit? In fact, humanity is doing so since thousands of years by breeding animals and cultivating plants for their own demands. However, transferring a directed evolution approach to the laboratory could raise concerns in the general public. We wanted to discuss our directed evolution approach with experts in the field of ethics. Hence, we talked to Prof. Dr. Philipp Stoellger from the theological department of Heidelberg University. Prof. Stoellger pointed out that he, as a theologist, has no general concerns about intervening in evolution and harnessing its potential for human benefits as long as the experiments are kept under strict laboratory conditions. However, ethical questions arise, if powerful and efficient techniques like PREDCEL and PACE are misused for military or otherwise harmful purposes or if modified organisms are released into the natural ecosystem. It is the duty of each and every scientist to reflect about the consequences of their research. Could this new discovery or invention be misused in any way? Which safety measures and precautions can be taken to prevent misuse? To face ethical concerns about directed evolution approaches, Prof. Stoellger recommended to firmly integrate professionals from other fields into such substantial projects, like environmentalists, human rights activist, ethics commissions, artists and politicians. This is reasonable because only an interdisciplinary group of professionals can make decisions with consequences affecting all humanity. Prof Stoellger was also impressed about the concept of iGEM and how this competition in Synthetic Biology encourages young scientists to reflect on safety, environment and the involvement and contribution of the general public to the design and elaboration of the scientific project. Following this concept, young scientists would grow to become responsible researchers in the future.