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.

Team:UCopenhagen/Human Practices

Introduction