Team:UCopenhagen/Human Practices
Introduction
We have aimed towards incorporating Human Practice-thinking into as many aspects and phases of our work as possible. Therefore we did already in the choosing of our project decide some factors that we found important to take into consideration when choosing our project: First and foremost we wanted to make something that was not going to interfere with nature or human bodies. We wanted to make a product that would make a positive difference in the world taking into account certain societal factors such as safety and usability, avoiding harm to the environment, and considering ethical downsides.
In order to find the right idea, we met up with different people and the team members spoke with their families, friends, peers and professors in order to find ideas on wich problems we could potentially solve with our project. We spent weeks discussing up-to-date topics such as microplastic pollution, radiation, antibiotic resistance, coculture and much more. You can read more about our choosing process here ….
We have also been concerned about safety at an early stage and has therefore contacted the Danish Center for Biopreparedness and biosafety in order to get their evaluation on our idea. Besides from that we had an extensive dialogue with the iGEM safety council and our internal safety officers. You can read more about their evaluation and our safety choices based on the feedback here ….
After choosing our project, we spoke with some different experts in the protein and space medicine field about which proteins would be relevant for space travel. We got a lot of interesting input and it had a big impact of how we now perceive the uses of our products and the problems it can potentially solve. You can read more about the experts and our integration of their input here …
When we were at Nordic iGEM conference in Lund, we noticed that we had a lot in common with the other Danish iGEM team, DTU Biobuilders. We decided to propose for a collaboration with DTU and at the same time Exeter asked us about collaborating. We ended up collaborating all three together. You can read more about our collaboration with DTU and Exeter here...
The (journey of) choosing our project
iGEM team copenhagen is a very mixed group of people, both in terms of age, gender, nationality and competencies - a diversity that we have embraced and taking into account by fx making a team contract that states expectations for team members. We were assembled in March and spend our first day together competing in building towers from spaghetti and marshmallows.
After the spaghetti we went straight to core values - a continues discussion taking place for several weeks. We decided that we wanted to make a product that was useful, and we wanted to investigate all potential downsides before making a choice - that was and is the most important values for all of us. We also wanted to have an almost flat team structure and to make a team contract in order to know what to expect from each other. You can read our team contract here…
We spoke with different people in order to learn about current problems that could be solved with synthetic biology. To get initial inspiration we met up with previous team members from iGEM teams at UCPH in order to discuss their projects and what kind of problems they were trying to solve. We asked friends, and family and got inspiration from different professors at the department for synthetic biology at UCPH.
At the end one meeting proved to be exceptionally fruitful for us, and that was the meeting with Astrobiologist, Lynn Rothschild. Rothschild has collaborated with previous iGEM Copenhagen teams as a supervisor for the iGEM team at Brown university. One early spring day this year she visited Copenhagen and found time to meet up with us and made a very interesting lecture about subjects such as upmass and self sufficiency. To make a long story short, Rothschild inspired us to explore problems in space further.
When one team member came into the office and had as an act of procrastination read about the Salmonella bacterium, the idea of using the injectisome to produce and release proteins was born. Many team members were immediately amazed with the idea, but we decided that we had to think it to an end before jumping into the lab.
Therefore we developed on three different ideas in parallel and did extensive research on societal impact of all of them. We learned a lot from our discussions of the three different ideas, and ended up deciding to choose the injectisome idea because it is not releasing gmo into nature, it is potentially beneficial both on earth and in space, it might in the future be a greener and more efficient method of producing protein, without taking any risks health wise or doing harm on any ecosystems.
(Pictures: Spaghetti teambuilding, Lynn + team)
Collaboration with DTU and Exeter
We noticed our common interests with DTU quite early. Already at the Nordic iGEM Conference in june, we were amazed by the DTU teams presentation and immediately felt the urge to work together. We spoke about how their ideas about using fungi for building on Mars, matched our idea about protein production on Mars - our common interest in exploring Mars was very obvious to us from the beginning. When we got home we decided to prepare ourselves for the proposal and contacted DTU to tell them how beneficial it would be to all of us if we worked together - come on, we're almost neighbors and we are working with the same topic?
In the meantime we got a request from Exeter asking if we would like to collaborate with them, and who could say no to that proposal? We couldn't, especially not since Exeter are working with a super interesting project about making oxygen with help from already present gasses and modified bacteria. Awesome, we thought. But what about DTU?
Luckily DTU said yes (or more accurately: "Yaaaas!!"), when we presented our idea of collaborating all three together.
When we decided to collaborate we didn't really have a plan. Brainstorming was a big part of our first Skype meetings, and because of our early start, our discussions managed to shape parts of each others Human Practice projects. We especially discussed how unusual it was for space related igem teams to question space travel ethically, even though there are lots of obvious questions to ask. We decided that we would like to explore the questions further and found especially the history of colonization, the arguments for and against Mars colonization and the ethics of colonization interesting and worthy of further exploration. We decided that we would make a report with the topic "Why colonize Mars?", where we would first analyze historical reasons for colonizing land, then analyze the main arguments in the public debate about Mars colonization and at the end discus our results in a ethical context.
We also contacted the Planetarium in Copenhagen in order to ask them for the possibility of giving a talk at one of their events, but instead of that they offered us a booth at one of the major cultural event nights in Copenhagen. We were very happy about that and contacted DTU immediately to hear if they would be interested in participating and having the booth together - Luckily they were very interested and we decided to extend our collaboration so it included the event at . You can read more about our collaboration at the Planetarium event here and find our report Here (link)
Choosing a protein
Integrated Human Practice
In parallel with developing our project we have been speaking with several experts within the field of health and space medicine. They have contributed with very valuable inputs for our project. One limit for choosing a protein for now is that protein therapeutics for improving the human conditions during space exploration isn't very established yet. Therefore, we have made a list of useful proteins that we theoretically should be able to produce with our system.
Proteins of interest for space exploration
IgG stan
IgG stan is a collection of IgG antibodies from human donors. The IgG stan is therefore a collection of antibodies with different specificity. IgG is very useful for presenting antigens to the immune system and can therefore function as a booster of the activity of the immune system of the host. Data shows that the immune system is dysregulated under microgravity and therefore it might be interesting to produce proteins that help improve the activity of the immunesystem [1]. We were pointed in this direction when speaking with Virginia Wotring.
Immune modulators
As mentioned above the immune system is believed to be dysregulated during microgravity [1]. Therefore, other proteins that affect the immunesystem could be interesting to produce with our system. This could both be cytokines, interleukines and other immune regulatory proteins. We were also pointed in this direction by Virginia Wotring.
Granulocyte Colony Stimulating Factor (G-CSF)
Radiation in space is markedly higher anda part of acute radiation syndrome is hematopoietic syndrome. This syndrome is characterized by a reduction in the number of neutrophils. Neutrophils are an important part of the innate immune system and as the number of neutrophils decrease the probability of infections increase. G-CSF promote the differentiation of neutrophils and can therefore be a possible countermeasure to low neutrophil cell count [2][3]. We were also pointed in this direction by Virginia Wotring.
Insulin like growth factor 1(IGF-1)
Has previously been addressed as a potential therapeutic protein to prevent the muscle atrophy [4]. The idea being that IGF-1 acts directly on the target tissue promoting growth. We had thought about producing IGF-1 ourself and discussed this with Jon Scott who thought that it might be very interesting and useful for long term missions to combine these anabolic proteins with exercise in space.
Growth hormone (GH)
Growth hormone has both long term and short term effects. The short term effects are diabetogenic while the long term effects promotes tissue growth especially muscle and bone. The long term effects of GH are mediated by IGF-1[5][6]. Therefore both IGF-1 and GH are very closely linked, and whether to use one instead of the other depends on the therapeutical context. We had thought about producing GH ourself but discussed it with Jon Scott who thought that it might be very interesting to combine these anabolic proteins with exercise in space.
Parathyroid hormone (PTH)
Parathyroid hormone is produced in parathyroid gland and is secreted in response to low blood calcium concentration. PTH activate the osteoclasts and thereby promote bone resorption and release of calcium and phosphate into the circulation. PTH also promotes secretion of phosphate in the kidney, thereby elevating the concentration of free calcium ions. Despite this PTH also has anabolic effects on bone and is currently used as a osteoporosis drug [7]which makes PTH a very interesting protein for us to produce.
Antibody/ single domain antibody
Almost all the experts we have talked to suggested us to produce antibodies, because antibodies are useful for a lot of different things. Our main idea as it is right now it to produce antibodies for detection of different molecules, this could be used both for differential diagnosis of astronauts but also for research purposes and help in a lot of different research purposes in space for instance enlightening what happens to the human body under microgravity. We have looked into which alternatives there is to normal antibody and found that there exists a single domain antibody that might be easier to produce and are as specific as the human one[8].
Requirements for production with our system
The principle on which our system works allows for extremely pure protein to be obtained, but at the other hand presents limitations concerning complexity of the proteins that can be produced. One limitation is folding of the proteins, once the protein of interest is secreted across the membrane it needs to be able to fold more or less by itself in the conditions of the buffer. This means that smaller and simpler proteins are better to produce with our system given that they are better at folding on their own. How big this folding challenge will be will become clearer when we have data on production of protein with our system. For now we have spoken with Michael Hecht at the European iGEM meetup who suggested we tried to produce his fusion proteins that were better at self-folding. If they work well we could try using his technique for protein production with our system and our protein of interest.
Because we produce our proteins using E. coli there are some limitations as to the posttranslational modification (PTM) of the proteins we intend to produce, for instance it is not possible to produce glycosylated proteins. PTM are more characteristic for eukaryotic systems, and this fact further complicates protein production, since some PTMs can't be performed in prokaryotic cells.
The protein to produce with our system should therefore be as simple as possible, able to fold by itself, have a minimum number of post-translational modifications and be relatively stable.
Based on these criteria we have tried to make a review of the feasibility of producing the proteins that might be interesting for space travel.
| Protein | Complexity | Structure (obtained from Uniprot) | Uniprot code |
| PTH | 66,3kDa
3 disulfide bonds, 4 glycosilations, 1 N-glycosilation |
Q03431 | |
| Camel antibody | 14,0 kDa
1 disulfide bond |
A2KD59 | |
| IGF-1 | 21,8 kDa
3 disulfide bonds |
P05019 | |
| G-CSF | 22,3 kDa
2 disulphide bonds and glycosylation |
P09919 | |
| Growth hormone | 2 disulfide bonds and 4 modified residues | P01241 | |
| Antibody (IgG) | 150 kDa (a), presence of disulfide bonds, too complex |
No matter which protein we choose to produce we will need to optimize the conditions. How important the theoretical limitations we present here are, will be further unveiled upon investigation and experiments.
To sum up, we propose to use our system for production of IGF-1, PTH, G-CSF and camelid antibody during long lasting space missions. This decision is first of all based on our interviews with experts within the field of space medicine. Based on their opinions we have further looked into how easy the different proteins would be to produce with our system (looking into PTM, size, the litterature etc). It has been very important for development of our project to have the possibility to speak with experts such as Virginia Wotring, Jon scott, Eva Horn Møller and Jørgen Sauer and we have very much incorporated their inputs into the very core of our project.
[1] B. E. Crucian et al., "Immune system dysregulation during spaceflight: Potential countermeasures for deep space exploration missions," Front. Immunol., vol. 9, no. JUN, pp. 1–21, 2018.
[2] E. Seedhouse, Space Radiation and Astronaut Safety. .
[3] M. P. Mac Manus, D. McCormick, A. Trimble, and W. P. Abram, "Value of granulocyte colony stimulating factor in radiotherapy induced neutropenia: Clinical and laboratory studies," Eur. J. Cancer, vol. 31, no. 3, pp. 302–307, Jan. 1995.
[4] Y.-H. Song, J. L. Song, P. Delafontaine, and M. P. Godard, "The therapeutic potential of IGF-I in skeletal muscle repair.," Trends Endocrinol. Metab., vol. 24, no. 6, pp. 310–9, Jun. 2013.
[5] C. S. Leach, N. M. Cintron, and J. M. Krauhs, "Metabolic changes observed in astronauts," J.Clin.Pharmacol., vol. 31, no. 0091–2700 (Print), pp. 921–927, 1991.
[6] W. Boron and E. Boulpaep, Medical Physiology. 2008.
[7] D. Aslan et al., "Mechanisms for the bone anabolic effect of parathyroid hormone treatment in humans," Scand. J. Clin. Lab. Invest., vol. 72, no. 1, pp. 14–22, Feb. 2012.
[8] M. M. Harmsen and H. J. De Haard, "Properties, production, and applications of camelid single-domain antibody fragments.," Appl. Microbiol. Biotechnol., vol. 77, no. 1, pp. 13–22, Nov. 2007.
Critical evaluation of our pure protein production system
We know that our product isn't the only way of purifying proteins. Therefore, we have tried to face the reality and find out how well our system perform compared to other production method. This evaluation is based on our very valuable internal discussions and what we have learned by speaking with experts.
Potential drawbacks
1.Efficiency
Our system probably can't come even close to current methods used in Pharmaceutical Industry, for example using E. coli or yeast pichia pestoris. It is not difficult to imagine huge bioreactors able to grow bacteria in volumes of broth up to few hundred litters at a time. Since we are limited with the surface of the membrane upon which our secretion system is dependent, it is not hard to imagine that in bioreactors where the whole volume is used for production of needed protein have higher yield. Even though we don't have exact numbers yet it is probably safe to assume that efficiency will be probably lower than of the current methods used in Pharmaceutical industry.
2.Size and complexity of the protein
Since proteins in our system are secreted in unfolded state there is a serious limitation in how big/complex the proteins can be. Only proteins that are able to fold on their own are appropriate for our system. It goes without saying that absence of PTM (post translational modifications) is almost a prerequisite.
3. Robustness of the process
Due to presence of highly delicate membrane which is crucial both for the integrity of the system (keeping bacteria from contaminating the injectable protein solution) and for the whole process of production of protein via secretion through it, this can be actually considered as "the weakest link in the chain". More work is required for characterising the properties of the membrane.
4.Presence of living potentially pathogenic bacteria
Since bacterial cells are not lysed (as in traditional protein production methods) the presence of living microorganisms - stepwise so close to the final product - this presents a certain risk, considerably higher than if the cell lysate had to undergo a number of purifications and analysis steps.
However there are also a few advantages of our system:
1. There is no other way to produce protein in space
Arguably they could produce it the "traditional way" – that is growing bacteria and then lysing it, isolating the protein from the lysate, purifying it etc… but that would be extremely difficult to do in absence of gravity and very hazardous in closed space of space station/spaceship. Also the amounts of water and chemicals for protein purification are prohibitively high for a space mission where water is scarce. The fact that astronauts are living in closed space with artificial circulation of air makes any release of potentially pathogenic bacteria very dangerous.
2.There is no "safer" way to produce protein in space
As mentioned above closed systems for handling bacteria are more desirable than "open" systems.
3.Purification steps are effectively eliminated
Protein is secreted in it pure form across the membrane thus eliminating expensive and labour intensive purifying steps.
4.Less biohazard waste produced
Some biohazard waste will inevitably be produced, but in considerably smaller quantities than via "usual" process of lysis and purification
5.No presence of lipopolysaccharides
Further improvement would be using E.coli strains with disabled lipopolysaccharide production, which could be another safety measure.
6.Produce what you need when you need and in quantities you need
Suppose a special protein for quite rare illnesses is needed. It is hard to expect that we will have a cure for every illness in the medicine kit onboard the spaceship. And even the medicines that we took from earth, what happens when we run out of them?
Conclusion
It is up to reader to make the judgment whether system seems promising in the context of space exploration or not. Considering that not much research has been done about medicine production in space, our system could potentially address several of the issues connected with "traditional" protein production in space. However due to extraordinary simplicity it could also find its uses on Earth.
Protein therapeutics
Therapeutics – just a different name for medicine. And protein therapeutics is just another type of medicine we use to save and prolong lives, just as for example antihypertensives or anticoagulants.
Figure 1: A structure of warfarin (perhaps the most famous anticoagulant).
Admittingly, proteins used in medicine are not that known, as for example warfarin, perhaps the most popular anticoagulant.
With exception of insulin of course. So what are protein therapeutics and what makes them so different from "normal" medicine?
Proteins are almost a completely new world when it comes to production, storage or administration to patients. Only 25 years ago a first recombinant protein therapeutic – human insulin – was introduced, yet today protein therapy is one of the most developing fields in pharmacy (1) Despite the enormous efforts of scientists over more decades we are only just taking a peek into the full potential of this new class of medicine.
And we still have a lot to learn.
Medicine, how do you produce it? What is it?
In pharmacy we obtain different chemical compounds with healing properties from various sources. Early in history of medicine the plants were the most common source of remedies – a term chemical synthesis was not so well defined back then. Of course people kept experimenting and gradually building up knowledge until reaching the level where we didn't have to rely solely on experience i.e. that plant killed the neighbour when he cooked it, but the one with yellow flowers healed the my cousin's cough.
It took a lot of time to figure out "what" was the thing that healed. One of the pioneers on that field was Paul Ehrlich which made the term "Magic Bullet" popular. In his opinion there must be a chemical compound that would selectively kill the bacteria yet not harm the human tissue. Inspired by colouring agents used to stain bacteria for microscopy he soon developed Salvarsan for treating Syphilis. It was one of the most widely prescribed drug after being put on market in 1910 and until production of penicillin in 1940.
To sum it up, medicine or API (active pharmaceutical ingredient), the compound is the one that does the "healing". It all comes down to chemistry. Even in remedies deriving from the plants, for example to cure malaria – it is a chemical "quinine" that kills the parasite plasmodium falciparum. (Bark from the cinchona tree was historically used as antimalarial agent, since it contained quinine).
Figure 2: Ehrlich Paul.
Many API are nowadays chemically synthesized in labs, strict regulations, careful handling and constant testings allow us to always have a safe pill in the pharmacy whenever we feel sick… and at low cost. What more could we possibly want?
Figure 3: A structure of quinine.
Figure 4: Exercise from Pharmaceutical Chemistry, a simple distillation, in one of the laboratories of Faculty of Pharmacy, Ljubljana. Courtesy of Rok Kelher.
How about proteins then?
It is slightly more complicated. The mechanism of action for previously described compounds can be really simple. Sometimes it is a matter of whether the compound can fit into a gap or hole in the enzyme. If it fits correctly it can slow down or accelerate it, and thus affecting the patient via changed metabolism or similar.
With proteins however, one needs to bear in mind that we are made of proteins. They are usually much, much bigger than the "traditional chemical compounds" and quite often extremely specific.
In order to treat patients we can either supply the patient with protein that he is unable to produce (for example insulin), develop immune protection of the patient (vaccination) or target "special targets" in the body. The following classification may shed some light on the whole matter: (1)
Group I: protein therapeutics with enzymatic or regulatory activity
• Ia: Replacing a protein that is deficient or abnormal.
• Ib: Augmenting an existing pathway.
• Ic: Providing a novel function or activity.
Group II : protein therapeutics with special targeting activity
• IIa: Interfering with a molecule or organism.
• IIb: Delivering other compounds or proteins.
Group III : protein vaccines
• IIIa: Protecting against a deleterious foreign agent.
• IIIb: Treating an autoimmune disease.
• IIIc: Treating cancer.
The following classification was taken from the article "Protein therapeutics: a summary and pharmacological classification" by Benjamin Leader, Quentin J. Baca and David E. Golan, published in Nature Reviews 2008. For further info see the references.
There is the sheer amount of different proteins that we could theoretically produce – current estimation is that there are 30,000 – 40,000 different genes in human genome (2), resulting in much higher number of possible proteins. However, there are few other characteristic that sets them apart from small molecule drugs. They are generally extremely difficult to produce, costly and unstable.
Production
For example it often requires genetical engineering, usually bacteria or yeast. By changing their DNA we trick them into producing the proteins we want. Afterwards they need to be somehow isolated and purified so that no other hazardous remains of the previous hosts would inflict injuries to the patients. Purification is therefore a very important step in pharmaceutical industry.
Cost
It is quite obvious that running a bioreactor, growing bacteria, purifying the protein and handling it with extreme care in sterile conditions is somewhat more expensive then running a chemical reaction even if it consists of multiple steps.
Administration to the patient
Perhaps you are familiar with how the insulin is administered. There are no insulin pills because being a protein it would get digested in our stomach (although scientists are busy trying to change that, for example Novo Nordisk was working on "oral insulin" (3). The protein therapeutics still have to be injected, which causes discomfort to the patient. Injection is not where the problems stop however. Many factors such as distribution through the body, degradation and clearance play an important factor in making things more complicated.
Stability
An area of intensive research. Depending on the protein, they may prefer sticking together instead of remaining dissolved in the water. So called aggregation or fibrillation is a nightmare of every scientist working in protein production. Insoluble aggregates are simply useless for the patient, not to mention that smaller so called subvisible particles are often immunogenic. (4)
Conclusion
The proteins offer a lot, yet they are also demanding. They are probably never going to replace traditional, small molecule medicine synthesised in chemical laboratories because of huge difference in cost. Nevertheless they will continue to address number of illnesses that would otherwise be incurable as we discover more and more protein therapeutics. One of the most interesting and promising areas is definitely cancer research.
References
1. Benjamin Leader, Quentin J. et al. Protein therapeutics: a summary and pharmacological classification. Nature Reviews, vol 7, January 2008
2. Lander, E. S. et al. Initial sequencing and analysis of
the human genome. Nature 409, 860–921 (2001).
3. https://www.novonordisk.com/about-novo-nordisk/perspectives/the-pursuit-of-oral-insulin.html
4. Subvisible Particle Content, Formulation, and Dose of an Erythropoietin Peptide Mimetic Product Are Associated With Severe Adverse Postmarketing Events., DOI: 10.1016/S0022-3549(15)00180-X
Human health challenges during space exploration
The human body is very complex and intricate and therefore the effect on the human body of being in outer space isn't trivial, and might very well vary depending on each person. Given that relatively few people have been sent to space, the sample size is small and therefore the full story hardly unveiled yet.
Radiation and DNA damage
On earth the magnetic field and the ozone layer protects us from most of the radiation. Therefore, the radiation level is even higher in space. For example, astronauts on the international space station receives over ten times the radiation than what's naturally occurring on Earth [1]. Radiation is dangerous because it introduces mutations into the DNA, and if these mutations aren't repaired in the right way we get mutations in the DNA that are permanent. This presents a great risk for developing cancer when being in space and receiving high levels of radiation for a long period of time. Radiation can also cause other diseases such as radiation sickness [1]. The high radiation levels might also result in hematopoietic syndrome, which is characterized by a reduction in the number of neutrophils.
Muscle loss
During low gravity the muscles aren't used as much as on earth and therefore the muscles weakens and muscle mass declines [2]. Countermeasures right now is exercise. The effect of exercise is good but doesn't work for all astronauts, and presents some requirements that might not be possible to have during long term mission such as a mars mission.
Bone loss
Low gravity also results in bone loss, and NASA has learned that without gravity working on the human body, bones lose minerals, with a density dropping at over 1% per month. By comparison, the rate of bone loss for elderly men and women on Earth is from 1% to 1.5% per year [1].
Kidney stones (closely connected to bone loss)
The excess of calcium in blood due to bone degradation has to be somehow secreted from the body. The condition is known as hypercalciuria, and it means that kidneys are desperately trying to retain the calcium homeostasis by increasing calcium secretion into urine. However by doing so they increase the risk of developing kidney stones. Supplements such as potassium citrate are taken to reduce that risk. [6][7]
Vision impairment
Some astronauts have experienced vision impairments after being sent to space, the reason for these impairments seem to be related to the fluid shifts that follows during low gravity. This fluid shift results in an increased intracranial pressure[3][5].
Immune System dysregulation
Dysregulation of the immune system has been reported during space mission where increased incidence of congestion, increased incidence of allergies and reactivation of previously latent herpes virus has been indicators of an altered immune system [4].
[1] K. Mars, "The Human Body in Space," 2016. [Online]. Available: https://www.nasa.gov/hrp/bodyinspace. [Accessed: 22-Sep-2018].
[2] L. B. Johnson, "NASA INFORMATION National Aeronautics and Space Administration."
[3] M. B. Stenger et al., "Risk of Spaceflight Associated Neuro-ocular Syndrome (SANS)."
[4] B. E. Crucian et al., "Immune system dysregulation during spaceflight: Potential countermeasures for deep space exploration missions," Front. Immunol., vol. 9, no. JUN, pp. 1–21, 2018.
[5] https://www.nasa.gov/mission_pages/station/research/experiments/1038.html
[6] https://www.nasa.gov/mission_pages/station/research/news/Strong_Bones.html
[7] https://www.nasa.gov/mission_pages/station/research/experiments/1018.html