Team:Hamburg/HP/Gold Integrated

Integrated Practices

About biosaftety & biosecurity: How to handle security relevant research and diminish the potential of abuse

Working with genetically modified organisms (GMO) bears great responsibility, working with GMO risk group 2 even more. That is why got us informed about the rules for working with organisms and genes risk group 2 stated by the iGEM Foundation on their biosecurity website and got in contact with the iGEM Safety Committee even before planning our biobricks or the practical work in the laboratory. In addition to that, we look up all applying legal rules of Hamburg University, the federal country of Hamburg and the federal republic of Germany, to really take all laws and rules into account. We gathered all the information and went through the planning of the whole project, together with Professor Doctor rer. nat. Heisig (Biochemical Institute, University of Hamburg), supervisor in charge for the S2-laboratory, and Professor Doctor med. Aepfelbacher (university medical centre Eppendorf, Hamburg, Institute for Microbiology, Virology and Hygiene) a specialist in the field of microbiology, virology and infectious disease epidemiology. Professor Aepfelbacher significantly contributed to our safety precautions concerning the work with the Yersiniabactin gene as well as counselling us regarding other safety measures in the lab. To be able to start the research with the chosen genes and the chosen security level we had to apply for an approval at the concerned department of Hamburg in advance. The approval was granted and only afterwards we were allowed to start our work. But beside the laws and rules – what does it mean to work with risk group 2 organisms? How can we handle our knowledge in a responsible way and simultaneously follow the rules of scientific practices (and thus the rules of iGEM)?

To resolve these questions and the security implications of our planned work, we invited Doctor rer. nat. Mirko Himmel from the Carl Friedrich von Weizsäcker Centre for Science and Peace Studies (ZNF) of Hamburg University, from the Research Unit for Biological Weapons and Arms Control. He gave a lecture on “Ethics and Law in Research and Handling safety relevant Work” and afterwards took some time out to talk with us about our project and give us advice.

One focus was on the Dual Use Research of Concern (DURC; especially concerning science with dual purposes). DURC are scientific research works e.g. in life sciences, which aim to create something good, but can be abused to intentionally harm. There are experts, who generally would include all researches in infection medicine and toxicology. Basically, our project falls into this category of DURC, too. Of course, our aim is, to achieve something good with our gallium loaded siderophores as saving lives through a new cure for multiresistant germs in hospitals. Still we potentially show the way to a new bioweapon use of the used GMO through the single steps of our research. We’d never have something like this on our agenda and deeply condemn the use of bioweapons, but the possibility of abuse is there. Therefore, it is highly important to secure our research and possible outcomes against abusive use in advance.

DURC experiments feature the deliberate raise in toxicity of biological agents (e.g. bacteria, viruses, or toxins) or in virulence e.g. as a pathogen might (easier) spread by improved droplet infection. Also, researches are included into the DURC, which lead to an expansion of host or cell specificity (the bandwidth of possibly effected species or types of cells), experiments, which could result in the inefficacy of a vaccine and/or antibiotic, bypass a diagnostic procedure as well as procedures which lead to an altered or improved antibiotic resistance.

virulence, antibiotic resistance and the sensitivity to antibodies, as well as change or nullify the effectiveness of diagnostic procedures. The efficiency of diagnostics and the antibodies are affected by the same genetically determined change: For the induction of the synthesis we must add salicylate into the cultivation medium. By doing this, we also change the protein surface of the bacteria (E. coli K-12). This includes the antibody binding sites, optical traits and the expulsion of pigment. Our risk assessment revealed, that this effect is insignificant, as this reaction will only occur transient within the bioreactor after the addition of the substrate. The physiological modification is well researched and can be exactly rechecked. The same applies for antibiotic resistances: The resistance against many common antibiotics increases by the depletion of the ingestion channels and the raised gene expression of efflux pumps, but only as long as salicylate is added. A stable change of E. coli K-12 traits in a natural surrounding without a sufficient selection pressure is not possible. In addition, E. coli K-12 lacks the ability to populate the intestine. A survival in natural surrounding is therefore out of question.

The potentially increased virulence of the generated GMO, in our opinion, derives from the siderophore itself (as it is e.g. part of the high pathogenity island von Y. pestis) and its ingestion channels, which can lead to a heightened proliferation within the host organism. The latter only contribute in a passive way, as in the presence of other siderophore creator ingestion channels are added in their siderophores, too. The siderophores themselves raise their virulence, as soon as corresponding ingestion channels are available. Yersiniabactin, out of our two siderophores, is the more important, since Pyochelin is not produced during infections with facultative pathogenic bacteria. In addition, Pyochelin has a low affinity to melatonin and is very ineeficient within the human body. Pyochelin only is a selection advantage for Pseudomonas aeruginosa in its native milieu. Yersiniabactin on the other hand is an important pathogenic factor because it enhances the virulence of pathogens within the lungs. Yersiniabactin within the serum is bound by certain proteins and therefor inactivated. Since there is no direct contact with the blood serum within the lungs, especially facultative pathogenic bacteria, which can cause pneumonia, display Yersiniabactin.

Subsequently, Mirko Himmel discussed with us the possibilities we have with this knowledge, to design our biobricks as safe as we can, and which additional security measures wen can take to keep the endangerment by e.g. increased virulence as low as possible. As one of the precautions we spatially separated the genes for the development of the siderophores from the transporters, as well for the inner as for the outer cellular membrane on the plasmids. They are never aggregated on the same plasmid. Furthermore, we never insert the mentioned different plasmids into one mutual host organism. The genes for the transporter proteins are accordingly only transformed within the test stem and the genes needed for the synthesis of the siderophores are only brought to expression within the producer. As a result, the alteration of bacteria is only evoked within the producer and never in the test stem.

In addition to that Mirko Himmel strongly advised us to construct the bacteria and plasmids not to survive in the laboratory environment. At first, we thought about inserting a kill switch. This confronted us with the problem, that both plasmids already had a length of more than 10,000 base pairs, still the idea was noted down for later designs. An alternative solution was the selection of a bacteria stem with a known genetic defect in the synthesis of amino acids. This is the case with E. coli BL21, a tryptophan synthesis deficient stem, which belongs to the risk group 1 but without affording specific security measures (see ZKBS directory for E. coli stems). These bacteria need the tryptophan synthesis way, respectively the intermediate stages Chorismat and Isochorismat. This is want we want to utilise and adjust it to our siderophores Pychelin and Yersiniabactin. We intended to cultivate our final products in the cysteine synthesis deficient stem E. coli JW1267-1. Cysteine is the basis of both siderophores and all involved proteins and within the E. coli JW1267-1 stem would lack the possibility to produce siderophores.

In the end, we went through all safety considerations in advance and realised all proposals from Mirko Himmel and by this also fulfilled all safety constrains by iGEM and the German departments. With the chosen approach we utilise the principal of a so called biological safety measure, consisting of host organism and vector (plasmid) classified as safe. The virulence genes Pyochelin and Yersiniabactin in this way of application don’t pose a danger above security level 2. The level of potential abuse was minimised as far as possible by the described design. We never shared any detailed information on social media and made sure that no one, beside our own group, supervisors and the concerned department got hold on any safety implications of our project, because the simple knowledge can be a safety relevant resource. All members of our iGEM Team were involved in the process of evolving safety measures and abuse safekeeping and now have a much higher awareness of handling DURC and the application of safety measures. We also had a final meeting with Mirko Himmel at the end of our project for a final risk assesment, before handing in the results into the iGEM contest.

Mirko Himmel – Interview integrated human practices

Interview

Hello Mirko Himmel, the last time we met we spoke about biosafety.

Since our readers don’t know you, would you please introduce yourself:

I am biochemist by training. I worked several years in several basic research labs, my last place was the Institute for Medical Microbiology, Virology and Hygiene at the University Medical Hospital Centre Eppendorf here in Hamburg. Currently, I am working at the Centre for Science and Peace Research at the University Hamburg and I am specialized in biological arms control and biosecurity matters. Besides that, I am working at the Department for Microbiology and Biotechnology on projects involving bacteria such as Bacillus thuringiensis as well as the pathogenic Bacterium burkholderia.

We work with synthetic biology. Why is biosafety and why is biosecurity an important issue?

Synthetic biology aims at the introduction of new, engineered biological synthesis pathways by the use of very selective, focused genetic engineering. One work horse would be the use of genetically modified organisms (GMO) which will show the novel, artificially introduced elements, called sometimes “BioBricks”. That are (at least in theory) fully understood, well described biologically active elements such as expression genes, selection makers or controllable promoters which allow induced gene expression for example. Synthetic biology thrives at taking full control of biological process for the good. Examples would be the production of otherwise inaccessible chemical substances such as complex organic molecules essential curing diseases.

But if you start working with biological material/biological agents, very soon you want to be sure that all work is done in a safe manner. Nobody wants to get an infection by bacteria or viruses used in the genetic engineering lab. Therefore, a set of rules and ordinances have been developed over the last decades which support scientists and co-workers to perform their experiments in a safe way. Biosafety regulations and biosafety measures help us in our daily work and every employee (and even lab guest!) have to be introduced to them BEFORE entering the lab and start the work.

But there is also another side of doing work with biological agents, especially, if there are pathogenic or can cause an intoxication. Other people, seeking to cause harm by using biological material, can use make use of our agents you are working with or the knowledge you obtained or even the equipment you are using. Who would that be? Think of bioterrorists for example who want to spread terror using disease-causing biological agents. But theft might also be a problem. “Biocrime” is a term which refers to the misuse of biological stuff to cause harm or kill people on the level of an individual perpetuating individual criminal goals. Or think of states interested in the development of weapons of mass destruction (WMD). That could be nuclear weapons, chemical weapons or … biological weapons! That had happened in the past already! So, we know from history, that state-driven biological warfare programs potentially could reach a high level of sophistication. The Biological and Toxin Weapons Convention (BWC in short) was put into force in 1975 and prohibits the development, production, stockpiling etc. of biological and toxin weapons (BW) and requested member states to completely destroy potentially existing BW stockpiles as well as the destruction/conversion of development, production, storage and testing facilities used for offensive purposes. So, also in Germany being one of the 178 members of the BWC the development of biological weapons is forbidden by national law! But in the past, there were also illicit offensive BW activities, e.g. in the former Soviet Union or in Iraq. In this respect, also some kind of knowledge transfer took place during the Cold War allegedly involving also Egypt, Syria, North Korea, China or Iran. No hard proofs there in the public domain so far as I know. But from the lessons learned about the Iraqi BW program, more and more non-proliferation activities were put in place which aim at the mitigation of any transfer (proliferation) of materials, technologies, equipment as well as knowledge usable for the development of WMD. One example is UN Security Council Resolution 1540 issued in 2004 calling all UN member states to implement appropriate proliferation counter-measures.

Today, we term measure aiming at the mitigation of misuse of biological material and scientific knowledge as biosecurity measures. Biosecurity is not thought to impair science, but to allow scientific work taking place in safe and secure working environment! And there is some good news: many prerequisites for a robust biosecurity regime are already covered by biosafety measures anyway in place - if these measures are comprehensively implemented and obeyed.

And how you can help us setting up required biosafety and biosecurity measures?

Most importantly, you have to learn about the German legislation dealing with biosafety (“Biostoff-Verordnung”, “Gentechniksicherheitsverordnung”,” Technische Regeln für Biologische Arbeitsstoffe”, „Infektionsschutzgesetz” and so on). But I learned know from you that you already are in the picture. You seem to be in touch with all the right people at the university, who are responsible for giving you advice and who should supervise the work you are planning to do. Biosafety measures are important to protect yourself, your coworkers and the environment from biological hazards. One important pillar is to conduct the mandatory risk assessments BEFORE you start your work. It is of great importance getting all required official licenses by the local authorities in advance. Usually, you will get great support by the authorities in the planning phase, because they want to support progression of science.

The rules of the iGEM competition demand from any project team the awareness and the will to think about safety AND security implications of the planned work and … very importantly of the “products” of your work. One cannot ignore the responsibility of all of your iGEM team members for the experiments and the results! You have to take the responsibility and do everything you can in order to (a) set up a really fancy iGEM project … but on the same moment (b) do a proper risk assessment and do everything to minimize potential misuse of your work. The latter might ring a bell: it is the biosecurity aspect of your work I am speaking about now.

Therefore, you have to know about the (many, many) good things, but also about the bad things about science. In my talk, I presented to you the historical context of biological warfare, the misuse of biology which once happened to fulfill hostile purposes. And there might be still nowadays actors who are very interested in the knowledge and products you obtained. Therefore, you have to restrict access to your lab space, you have to restrict access to your lab journals and technical background papers, and you should not spread knowledge about your project in great details. Especially, when you use information channels where you do not have control about the flow of information.

There are technical barriers which you can implement in your project in order to prevent misuse. The iGEM rules give you an orientation here, what would be important to think about (one key word here: genes encoding toxins. Are you allowed to work with them? Is it really necessary to work with them? Can you assure that nobody would make hostile use of them once you finished your project?). We already discussed your project plans and found some promising ways to implement effective safety and security barriers. That is, because on the one hand your project is challenging and involves design concepts showing a certain dual use potential, on the other hand there are experimental steps which easily allow the integration of certain, well accepted security barriers.

To sum that up: Ideally, biosecurity measures are founded on the basis of appropriate biosafety measures and encompass both additional theoretical and practical steps to prevent the misuse of biology. You did already the right steps to implement biosafety as well as biosecurity measures. Our discussion about details of your project resulted in a well-balanced biosecurity concept.

What is dual use research of concern? How does it affect our plans?

The term “dual use” means, that you can use s.th. – in our case scientific achievements - for the good, but also for the bad. We already touched the topic “misuse of science for hostile purposes” several times now. To make it clear again: Many aspects of biological sciences show a dual use potential: biological materials you are working with, equipment and technical information you use, knowledge you use or produce by yourselves. But there is a category of research endeavors which go far beyond the typical dual use potential found in the sciences: dual use research of concern. What is meant by “concern”? There are some types of experiments which would result in biological agents (or knowledge, technical equipment etc.) which are much more readily usable for non-peaceful purposes than the those resulting of the fast majority of experiments in the biological sciences. In other words, there are different risk levels of misuse of science associated with certain types of experiments. One possibility to categorize these types of experiments are presented along with the report of the Fink committee as I explained to you in my talk.

In your case there are several dual use aspects of your planned project: the biological agents used show some rather low misuse potential, but the genes/gene products you are plan to work with can at least in theory interfere with essential human body functions such as proper immune defense and the uptake of iron ions. Yersiniabactin genes are found in Yersinia species with Y. pestis being one of the well-established biological warfare agents of past offensive BW programs. BUT, that does not necessarily mean that any work on Yersiniabactin is forbidden now by international and national law or that research and development activities on that protein will only fulfil malicious purposes. That is by no means true! It is a matter of intent and the context of the work done. But in order to hamper any attempts of misusing scientific work on Yersiniabactin it is essential to perform a proper risk assessment. That must be done before the planned work will start. The risk assessment then should result in the implementation in all required biosafety and biosecurity measures well in advance before practical work will start.

In sum, following the iGEM rules, international (BWC!) and national regulations, good laboratory practice, good scientific practice as well as fundamental ethical principles (!!) you can conduct your project in a safe and secure manner. But you have to take the responsibility as a scientist.

We introduced our project plans to you. In your impression, are there any specific parts of our project that are of specific concern?

Interference with iron uptake in the human body in the course of counter-acting bacterial infections is challenging! You may target both, the pathogen as well as the host organism! Therefore, a carefully developed prevention strategy to minimize biological hazards is mandatory. One aspect would be the induction of multiple resistance against conventional antibiotics. In your case we are talking about standard antibiotics used in the laboratory, but not in the clinics. But anyhow, that is one thing you should keep in mind: altering the GMO’s susceptibility against antibiotics. Another security aspect is the intended interference with the iron uptake by those pathogens you are aiming at. You load your siderophores with gallium (Ga), fair enough, clever strategy. But what do you know about the amount of Ga which would be transferred into the human body once your therapeutic approach would be used under real world conditions? Any Ga intoxication possible? Think of elder patients having a lot of other malfunctions of their body! Is your method safe? Another issue would be the coexistence of several bacterial siderophores within the human body. Is there any possibility that other facultative pathogenic or even usually non-pathogenic microorganisms would re-use/”misuse” your strategy to survive within the host? That is a matter of different uptake strategies employed by different bacterial species and one should test that in the lab first, before you go live with your therapeutic approach. And of course, interference with ion (!) uptake per se could be an interesting strategy also for those actors looking for s.th. which might cause harm in humans, animals or plants.

For our safety assessment, we worked closely with Professor Heisig and Professor Aepfelbacher to receive an official license to conduct our experiments. We introduced our safety measures to you. What is your assessment regarding Dual Use Research of Concern? Are our measures sufficient?

So far as I can see it: yes! All what you have told me make clear that you did the right steps in the right sequence. First, you developed a challenging project which aims at a novel, fascinating therapeutic strategy to fight against nasty bugs (i.e., infectious disease-causing pathogens) by using a rather simple but theoretically quite effective method. But then, you were not just starting the experiments and look what might happen. No, you then went back to the desk and did a reasonable, comprehensive risk assessment involving the input from many experts. From our discussions, I learned that you are well aware of the promises but also the potential risks of your project. You did the required considerations well in advance – that is exactly what Good Scientific Practice and responsible conduct of science demands! You told me that you studied relevant literature and figured out what others have already published about the several aspects of your project (e.g., induction of antibiotic resistance, problems caused by iron depletion in the human body, work with Yersinia-derived genetic information). Once you have had reviewed the expert feedback and the information obtained from the literature you were re-writing some details of your project plans in order to achieve a maximum of biosafety and biosecurity precautions. In my view, your measures are sufficient. Especially, because we are not talking about large-scale production of the GMO generated in the course of your project nor clinical trials involving test with patients in a hospital environment. In these cases, there would be much more to think about. But that is another story to tell…!

To conclude the interview, what is your general assessment, we spoke twice about biosafety and biosecurity issues. In between our meetings we had time to implement your suggestions from our first meeting. Were we successful?

I am quite confident that you are conducting your iGEM project with a responsible and wholehearted attitude. You successfully performed the mandatory risk assessments and in addition learned a lot about biosafety and biosecurity. It was a great pleasure for me to guide you through the world of dual use research of concern and biosecurity issues. Together, we addressed the relevant points affecting your project and further developed your risk assessment. And last but not least: You are now practitioners in biosecurity! Congratulations from my side and all the best for the iGEM 2017 Hamburg University team!

Rolf Müller as a guest speaker

To get one more professional opinion on our project design we invited Prof. Dr. Rolf Müller the director of the Helmholtz Centre for Pharmaceutical research. The Helmholtz Centre for Pharmaceutical research was funded by the Helmholtz Centre for Infection Research (HZI) in Braunschweig and the Saarland University, combining expertise from infection research and pharmaceutical sciences.

Prof. Dr. Müller is leading the department of microbial natural compounds and coordinates the research field “new antibiotics”.

After some trouble finding the chemistry department Prof. Müller arrived and started with a talk presenting us his field of study, talking about “Alternative antibiotics from Microbes”.

As most antibiotics were developed in the 50s and 60s they are mostly of natural origin. In the last 30 years were no new antibiotics developed. Firstly because it was assumed that no more new antibiotics were required. Now we know it is only a question when a resistance against new antibiotics will appear, not if.

The reasons for the pharmaceutical industry not to develop new antibiotics are that the completed drugs are almost non-profitable and the requirements are extremely high. There can't be any side effects, it has to be low priced and is applicated only once for two weeks.

2

In Prof. Müllers department the research focus lies on finding new antibiotic compounds which stem from nature compounds. Those are best produced by ground living microorganisms (MOs) and fungi. One of those MOs are myxobacteria, microorganisms which are able to kill other microorganisms. They are ubiquitary, displacing other MOs and can be recognized by their typical fruit bodies. The usual method to find new possible targets is to screen the genomes for a frequency of resistance, meaning it is analysed what has changed in the genome after gaining the resistance. With those approaches new possible antibiotics should be identified and analyzed.

3

After the talk we briefly presented our project idea and engaged in a meaningful discussion about pathway design and non-ribosomal-pathway design. During the discussion Rolf Müller pointed out that our design already was pretty good but had some additions to make. All in all we got great feedback about our project design.

Another focus of Prof. Müllers research group is infact the development of non-ribosomal-pathways, which is exactly what we were doing. He complimented us for our constructed pathway but pointed out that we were missing one important enzyme. The surfactin-synthetase-activating enzyme (sfp), which is responsible for activating the synthetases for the siderophores and essential for our project. As the native Phosphopantetheinyltransferases of our E.coli strains are not effective enough to activate overexpressed synthetases like HMWP2. On this important indication we included sfp into our part design, without which the siderophore synthesis would not be possible.

Prof. Müllers visit was a complete success for us by getting this valuable feedback for our project design and the hint what we were still missing.


×

Loading ...