Difference between revisions of "Team:UCopenhagen/Attributions"

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<h1>Bacteria</h1>
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<h1>Attributions</h1>
  
  
<p>
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<h2>Our Supervisors </h2>
Bacteria containing and actively expressing the injectisome system are pathogenic, meaning that they can make you sick. The system allows all kinds of different bacteria to shuttle disease-triggering proteins through the surface of our body and into our cells, which obviously makes you ill. The illness can vary somewhat from bacteria to bacteria, but the species that donated the injectisome-part we used - <em>enteropathogenic Escherichia coli (EPEC)</em> -  triggers diarrhea in humans, most prevalent in children(1).
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</p>
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<h1>A better version</h1>
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<p>
 
<p>
As such, the effects of that bacteria is unpleasant to say the least, and it's very much a must to to ensure the bacteria used in our Protein Printer is non-pathogenic. A system that produces pure disease-causing protein is near worthless, and the same can be said of a system that produces the target protein alongside disease-causing proteins. Our Protein Printer needs to produce only the wanted target protein, and nothing else for it to be functional.
+
All of our supervisors has been essential for our project. Without them, we wouldn't have been able to do anything.  They have helped with both the big and the small decisions and lead us in the right direction when we were in doubt. We are very thankful for the time and energy they have spent with us while our project has been growing. Thank you so much!
 
</p>
 
</p>
 
<p>
 
<p>
In order to ensure this, we acquired a bacterial strain with the help of Luis Ángel Fernández Herrero (PhD, Principal Investigator, National Center for Biotechnology (CNB) Madrid, Spain), that expressed the EPEC injectisome in the non-pathogenic E. Coli K-12 chassis.
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<strong>Associate Professor Sotirios Kampranis, </strong>Center for Synthetic Biology, University of Copenhagen
 
</p>
 
</p>
 
<p>
 
<p>
This means that the useful injectisome has been separated from the disease-causing parts of the bacteria and is now functional in a non-pathogenic bacteria instead. This new and improved - for our purposes at least - EPEC bacteria was dubbed <em>Synthetic injector E. coli (SIEC) </em>(2)
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<strong>Centre coordinator Nanna Heinz, </strong>Center for Synthetic Biology, University of Copenhagen
 
</p>
 
</p>
 
<p>
 
<p>
The new SIEC bacteria has the advantages of having the injectisome system, but is not capable of making the natural disease-causing proteins alongside it. This strain is therefore way more suitable for our purpose of producing proteins made for actual medical purposes without the fear of other diseases!
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<strong>Postdoc Simon Louis Théodore A Dusséaux, </strong>Center for Synthetic Biology, University of Copenhagen
 
</p>
 
</p>
<h1>The Injectisome</h1>
 
 
 
 
<p>
 
<p>
The injectisome (Type III secretion system) is a needle-like, protein structure used by some  pathogenic bacteria (in our case <em>E. coli</em>) to inject toxins into other cells, for example in our gut, to make the environment more habitable for pathogenesis. For example, Injectisome-producing <em>E. coli </em>uses the injectisome to attach itself to the intestinal cells and inject toxins into them to make them favourable for colonization. (1)
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<strong>PhD Fellow Morten Raadam, </strong>Center for Synthetic Biology, University of Copenhagen
 
</p>
 
</p>
 
<p>
 
<p>
The injectisome´s structure is in theory quite complex but simple to understand if one just thinks of a syringe, but a tiny molecular syringe. The base is made up of circle like structures which are both on the inside and outside of the cell membrane, to keep the injectisome in place. These circle like structures are connected together with a centrally located tube structure.  On top of this base comes the needle structure which, on its, top has a, so called, tip structure, which aids in attachment and forming of the hole which toxins are injected through. (2)
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<strong>PhD Fellow Davide Mancinotti, </strong>Center for Synthetic Biology, University of Copenhagen
 
</p>
 
</p>
 
<p>
 
<p>
<p style="text-align: right">
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<strong>PhD Fellow Cecilie Cetti Hansen, </strong>Center for Synthetic Biology, University of Copenhagen
17</p>
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</p>
 
</p>
 
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<p>
 
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<strong>PhD Fellow Victor Forman, </strong>Center for Synthetic Biology, University of Copenhagen
 
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</p>
<pre class="prettyprint">Injectifacts:
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<p>
-The Injectisome translocates more than 25 effector proteins, which make the infected cell habitable.
+
Furthermore we would like to give a special thanks to the inspiring people at the Section for Plant Biochemistry at University of Copenhagen for always helping us out and answering our questions.
-The EPEC injectisome in our strain is one of the biggest injectisome reaching up to 700 nm
+
</p>
-The SIEC strain we used can have a diameter of 0.68-1.0µm, which is about 70 times smaller than the thickness of your hair (3)
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<h2>Others</h2>
</pre>
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<p>
 
<p>
                                           
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<strong>Aaron Berliner</strong>, Graduate Student at Arkin Laboratory and NSF Fellow at UC Berkeley.  We were put into contact with Aaron through Adam Arkin and aaron was very nice and also helped find some interesting material from NASA regarding space medicine.
 
</p>
 
</p>
 
<p>
 
<p>
References:
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<strong>Adam P. Arkin, </strong>Professor and Director of The Center for the Utilization of Biological Engineering in Space (CUBES). Adam<strong> </strong>was very nice helped us in the right direction by answering our mails with questions regarding biotech development in the space industry. Adam was also very nice and forwarded our mail to other experts that we could be interested in speaking with. 
 
</p>
 
</p>
 
<p>
 
<p>
(1) Ochoa, T. J., Barletta, F., Contreras, C., & Mercado, E. (2008). New insights into the epidemiology of enteropathogenic Escherichia coli infection. <em>Transactions of the Royal Society of Tropical Medicine and Hygiene</em>, <em>102</em>(9), 852–856. https://doi.org/10.1016/j.trstmh.2008.03.017
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<strong>Alesandra Luchini</strong>, PhD, Postdoc at Lise Arleths group at the Niels Bohr Institute, University of Copenhagen. Alessandra met with a part of our wet lab team and helped brainstorm what artificial planar membrane-technology we could use in our experiments going forward.
</p><ul>
+
</p>
 
+
<li><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575077/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575077/</a></li></ul>
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+
 
<p>
 
<p>
(2) Ruano-Gallego, D., Álvarez, B., & Fernández, L. Á. (2015). Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells. <em>ACS Synthetic Biology</em>. <a href="https://doi.org/10.1021/acssynbio.5b00080">https://doi.org/10.1021/acssynbio.5b00080</a>
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<strong>Birger Lindberg Møller, </strong>Professor and Head of the Section for Synthetic Biology, University of Copenhagen. <strong> </strong>We really appreciate that, Professor Birger Lindberg Møller, as head of the section for synthetic biology, provided us with a letter of recommendation. This was useful for our funding applications.  
 
</p>
 
</p>
 
<p>
 
<p>
(3) Zaritsky A, Woldringh CL. Chromosome replication rate and cell shape in Escherichia coli: lack of coupling. J Bacteriol. 1978 Aug135(2):581-7<strong>PubMed ID</strong><a href="http://bionumbers.hms.harvard.edu/redirect.aspx?pbmid=355235&hlid=">355235</a>
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<strong>Eva Horn Møller, </strong>PhD,<strong> </strong>Senior Drug Product Specialist at Zealand Pharma A/S. We had a very fruitful discussion with Eva Horn Møller regarding our project. Eva stressed out the importance of having the patient and his safety in mind. She further pointed out that we need to take special care to determine what is secreted in our system  in addition to our protein of choice. She suggested that we could use a special <em>E. coli </em>strain with disabled lipopolysaccharide. These are all ideas that we intend to incorporate into our project both long term and short term. Eva also suggested that working on smaller, non-glycosylated peptides – including antibody fragments – may be the best way to go.
 
</p>
 
</p>
 
<p>
 
<p>
                                                   
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<strong>Lise Arleth</strong>, Professor in structural biophysics at the Niels Bohr Institute University of Copenhagen. She helped answer preliminary questions about artificial membranes, which helped guide the development of our membrane-focused experiments. She also put us in contact with Dr. Alessandra Luchini.
 
</p>
 
</p>
 
<p>
 
<p>
     
+
<strong>Luis Ángel Fernández Herrero</strong>, PhD, Principal Investigator, National Center for Biotechnology (CNB) Madrid, Spain. Luis sent us the bacterial strain used in all our experiments and for this we are very thankful.
 +
</p>
 +
<p>
 +
<strong>Jon Scott, </strong>PhD, Medical Projects Team Lead, Space Medicine Office, European Astronaut Centre. Jon told us that there is interest in combining exercise with anabolic drugs such as PTH, GH, IGF-1 in order to improve the effect of exercise. Furthermore, there are some drawbacks with exercise as it is right now such as, 1) it is time consuming, 2) not all astronauts like to do it, 3) it produces heat, CO<sub>2</sub> and moisture and 4) it increases the consumption of food. Jon also mentioned that proteins that could be used for diagnosis og for research purposes during space travel also would be very useful to produce with our system.
 +
</p>
 +
<p>
 +
<strong>Jacob Hofman-Bang, </strong>PhD, Senior Scientist, Centre for Biosecurity and Biopreparedness. We contacted Jacob in order to hear about the biosafety aspects of our project. Jacob made a very good analysis of our product and concluded that in the form it has right now there was not biosafety issues.
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
<strong>Jørgen Sauer, </strong>PhD, Fermentation Scientist from Novo Nordisk. Jørgen helped us envision the full application of our project in space missions and gave us quite a few advices. Being an enthusiastic scientist himself always open for bold and innovative ideas he quickly pointed out that it would have been even better if astronauts could produce the necessary DNA onboard and transform bacteria themselves! This way there would be almost no limit as to what protein they could produce! As we were concerned about how safe would be to actually use our system we went into discussion about what regulations do they abide by in industry. Keeping pH as close to the physiological as possible is a good idea if we want to prevent discomfort of the patient during injection, however stability of protein at that pH should also be kept in mind. FDA and other regulatory organs usually focus on amount of lipopolysaccharides present in the final product (amebocyte lysate test is routinely performed), which for our system shouldn't present a problem. The guidances concerning how pure should the final protein be are simple: as pure as possible! And if you cannot make it more than 99,9% pure than you need to. Further discussing a few interesting technical aspects of our design and its use in space (absence of gravity may make things really interesting) he pointed out that the big advantage of our system could be lower amount of biohazard waste produced in the process (especially if compared to how we produce and purify proteins on earth).
 +
</p>
 +
<p>
 +
<strong>Jane Nøhr</strong>, PhD, Cluster Manager at Biopeople, Denmark's Life Science Cluster. Jane provided us with very useful input both concerning the project and the current processes in the industry. Jane drew our attention to the current way of protein production in the industry using <em>Pichia pastoris. </em>Jane also suggested us to pay a special attention to yield and purity when developing our product.
 +
</p>
 +
<p>
 +
<strong>Kirsten Jørgensen</strong>, PhD, Associate Professor, Vice head of Department for teaching and Work Environment Coordinator at the Department of Plant and Environmental Sciences, University of Copenhagen. She assisted us greatly when filling out the Safety Form and ensuring we applied for the appropriate internal safety applications to ensure we were able to do our experiments. Furthermore, she provided us with a letter of recommendation that was useful for our funding applications.
 +
</p>
 +
<p>
 +
<strong>Karen McDonald, </strong>PhD, Professor of Chemical Engineering at the University of California at Davis, Institutional Co-I for CUBES. We got into contact with Karen through CUBES. We discussed the biomedical aspects of the proteins we want to produce for spacetravel with her and she gave us some very valuable inputs. Karen said that the 2,5 hours of exercise per day (which is the standard right now for astronauts) might be difficult for long term space explorations and therefore some drugs would potentially be very useful when facing this problem. Karen McDonald's research group are working on producing the protein drug PTH (parathyroid hormone) in a way that is suitable for space travel. Karen McDonald also mentioned anti-cancer drugs as something that would be very useful to produce  during long term space travel.
 +
</p>
 +
<p>
 +
<strong>Lonnie Grove, </strong>PhD, postdoc at<strong> </strong>The University of Copenhagen and Department of Orthopedic Surgery, University of California San Diego. Lonnie was one of the first experts we talked with and she help us understand the general health issues that arise during space travel. Lonnie said that production of antibodies would be useful for space travel.
 +
</p>
 +
<p>
 +
<strong>Lynn Rotschield, </strong>PhD, Senior Research Scientist, Bio and Bio-Inspired Technologies, Research and Technology Lead for NASA HQ STMD.<strong> </strong>Lynn visited us in Copenhagen in the spring. We had a very interesting and inspiring meeting, which among other reasons let us in the direction of a space project.
 +
</p>
 +
<p>
 +
<strong>Mette Galsgaard, </strong>PhD Fellow Department of Chemistry, The University of Copenhagen. <strong> </strong>Mette<strong> </strong>has taught us how to make liposomes and supported lipid bilayers. She also helped with designing our liposome experiment.
 +
</p>
 +
<p>
 +
<strong>Michael Hecht, </strong>Professor, Department of Chemistry, Princeton University.<strong> </strong>We met Michael at the European Meetup in Munich where Michael gave a talk. Following that we were very happy to talk with him, and he gave us some very nice input regarding protein folding and production.
 +
</p>
 +
<p>
 +
<strong>Nikos Hatzakis, </strong>Associate Professor, Department of Chemistry, The University of Copenhagen. Nikos helped us designing the liposome and supported lipid bilayer experiment and made it possible for us to carry out our experiments using confocal fluorescence microscopy by letting us use the microscopes in his laboratory.
 +
</p>
 +
<p>
 +
<strong>Piers Millet, </strong>PhD<strong>, </strong>Director of Safety and Security at iGEM and co-chairs iGEM's Safety Committee. Piers<strong> </strong>helped us with filling out the safety forms and pointed us in the direction of Jacob Hofman-Bang who helped us by making a biosafety analysis of our project. Piers also sent our safety questions to the Australia Group (safety council).
 +
</p>
 +
<p>
 +
<strong>Poul Erik Jensen, </strong>Professor, Head of <a href="https://cpsc.ku.dk/">Copenhagen Plant Science Center</a>, The University of Copenhagen. Professor Poul Erik Jensen has earlier been supervisor for the iGEM team from University of Copenhagen. He therefore kindly provided us a letter of recommendation which was helpful for our funding applications.
 +
</p>
 +
<p>
 +
<strong>Søren Schmidt-Rasmussen Nielsen, </strong>PhD Fellow, Department of Chemistry, The University of Copenhagen.<strong> </strong>Søren has taught us how to use the confocal fluorescence microscope. He also helped with the design the supported lipid bilayer experiment.
 +
</p>
 +
<p>
 +
<strong>Tomas Laursen, </strong>PhD, Postdoc at Section for Plant Biochemistry, University of Copenhagen.  Tomas helped with preparing the protocol for liposome preparation and supported lipid bilayers and lent us extrusion equipment.
 +
</p>
 +
<p>
 +
<strong>Virginia Wotring, </strong>PhD, Adjunct Associate Professor, Center for Space Medicine and Department of Pharmacology and Chemical Biology, Baylor College of Medicine. We had a very interesting talk with Virginia Wotring on skype, where we discussed several aspects of our project. We also got to discuss which protein would be relevant to produce from a pharmacist's point of view. Virginia suggested that we could look into production of IgG-stan (type of IgG antibody which is used therapeutically), antibodies, immune modulators (cytokines and interleukins) and G-CSF.
 
</p>
 
</p>
 
 
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Revision as of 11:50, 14 October 2018

Attributions

Our Supervisors

All of our supervisors has been essential for our project. Without them, we wouldn't have been able to do anything. They have helped with both the big and the small decisions and lead us in the right direction when we were in doubt. We are very thankful for the time and energy they have spent with us while our project has been growing. Thank you so much!

Associate Professor Sotirios Kampranis, Center for Synthetic Biology, University of Copenhagen

Centre coordinator Nanna Heinz, Center for Synthetic Biology, University of Copenhagen

Postdoc Simon Louis Théodore A Dusséaux, Center for Synthetic Biology, University of Copenhagen

PhD Fellow Morten Raadam, Center for Synthetic Biology, University of Copenhagen

PhD Fellow Davide Mancinotti, Center for Synthetic Biology, University of Copenhagen

PhD Fellow Cecilie Cetti Hansen, Center for Synthetic Biology, University of Copenhagen

PhD Fellow Victor Forman, Center for Synthetic Biology, University of Copenhagen

Furthermore we would like to give a special thanks to the inspiring people at the Section for Plant Biochemistry at University of Copenhagen for always helping us out and answering our questions.

Others

Aaron Berliner, Graduate Student at Arkin Laboratory and NSF Fellow at UC Berkeley. We were put into contact with Aaron through Adam Arkin and aaron was very nice and also helped find some interesting material from NASA regarding space medicine.

Adam P. Arkin, Professor and Director of The Center for the Utilization of Biological Engineering in Space (CUBES). Adam was very nice helped us in the right direction by answering our mails with questions regarding biotech development in the space industry. Adam was also very nice and forwarded our mail to other experts that we could be interested in speaking with.

Alesandra Luchini, PhD, Postdoc at Lise Arleths group at the Niels Bohr Institute, University of Copenhagen. Alessandra met with a part of our wet lab team and helped brainstorm what artificial planar membrane-technology we could use in our experiments going forward.

Birger Lindberg Møller, Professor and Head of the Section for Synthetic Biology, University of Copenhagen. We really appreciate that, Professor Birger Lindberg Møller, as head of the section for synthetic biology, provided us with a letter of recommendation. This was useful for our funding applications.

Eva Horn Møller, PhD, Senior Drug Product Specialist at Zealand Pharma A/S. We had a very fruitful discussion with Eva Horn Møller regarding our project. Eva stressed out the importance of having the patient and his safety in mind. She further pointed out that we need to take special care to determine what is secreted in our system in addition to our protein of choice. She suggested that we could use a special E. coli strain with disabled lipopolysaccharide. These are all ideas that we intend to incorporate into our project both long term and short term. Eva also suggested that working on smaller, non-glycosylated peptides – including antibody fragments – may be the best way to go.

Lise Arleth, Professor in structural biophysics at the Niels Bohr Institute University of Copenhagen. She helped answer preliminary questions about artificial membranes, which helped guide the development of our membrane-focused experiments. She also put us in contact with Dr. Alessandra Luchini.

Luis Ángel Fernández Herrero, PhD, Principal Investigator, National Center for Biotechnology (CNB) Madrid, Spain. Luis sent us the bacterial strain used in all our experiments and for this we are very thankful.

Jon Scott, PhD, Medical Projects Team Lead, Space Medicine Office, European Astronaut Centre. Jon told us that there is interest in combining exercise with anabolic drugs such as PTH, GH, IGF-1 in order to improve the effect of exercise. Furthermore, there are some drawbacks with exercise as it is right now such as, 1) it is time consuming, 2) not all astronauts like to do it, 3) it produces heat, CO2 and moisture and 4) it increases the consumption of food. Jon also mentioned that proteins that could be used for diagnosis og for research purposes during space travel also would be very useful to produce with our system.

Jacob Hofman-Bang, PhD, Senior Scientist, Centre for Biosecurity and Biopreparedness. We contacted Jacob in order to hear about the biosafety aspects of our project. Jacob made a very good analysis of our product and concluded that in the form it has right now there was not biosafety issues.

Jørgen Sauer, PhD, Fermentation Scientist from Novo Nordisk. Jørgen helped us envision the full application of our project in space missions and gave us quite a few advices. Being an enthusiastic scientist himself always open for bold and innovative ideas he quickly pointed out that it would have been even better if astronauts could produce the necessary DNA onboard and transform bacteria themselves! This way there would be almost no limit as to what protein they could produce! As we were concerned about how safe would be to actually use our system we went into discussion about what regulations do they abide by in industry. Keeping pH as close to the physiological as possible is a good idea if we want to prevent discomfort of the patient during injection, however stability of protein at that pH should also be kept in mind. FDA and other regulatory organs usually focus on amount of lipopolysaccharides present in the final product (amebocyte lysate test is routinely performed), which for our system shouldn't present a problem. The guidances concerning how pure should the final protein be are simple: as pure as possible! And if you cannot make it more than 99,9% pure than you need to. Further discussing a few interesting technical aspects of our design and its use in space (absence of gravity may make things really interesting) he pointed out that the big advantage of our system could be lower amount of biohazard waste produced in the process (especially if compared to how we produce and purify proteins on earth).

Jane Nøhr, PhD, Cluster Manager at Biopeople, Denmark's Life Science Cluster. Jane provided us with very useful input both concerning the project and the current processes in the industry. Jane drew our attention to the current way of protein production in the industry using Pichia pastoris. Jane also suggested us to pay a special attention to yield and purity when developing our product.

Kirsten Jørgensen, PhD, Associate Professor, Vice head of Department for teaching and Work Environment Coordinator at the Department of Plant and Environmental Sciences, University of Copenhagen. She assisted us greatly when filling out the Safety Form and ensuring we applied for the appropriate internal safety applications to ensure we were able to do our experiments. Furthermore, she provided us with a letter of recommendation that was useful for our funding applications.

Karen McDonald, PhD, Professor of Chemical Engineering at the University of California at Davis, Institutional Co-I for CUBES. We got into contact with Karen through CUBES. We discussed the biomedical aspects of the proteins we want to produce for spacetravel with her and she gave us some very valuable inputs. Karen said that the 2,5 hours of exercise per day (which is the standard right now for astronauts) might be difficult for long term space explorations and therefore some drugs would potentially be very useful when facing this problem. Karen McDonald's research group are working on producing the protein drug PTH (parathyroid hormone) in a way that is suitable for space travel. Karen McDonald also mentioned anti-cancer drugs as something that would be very useful to produce during long term space travel.

Lonnie Grove, PhD, postdoc at The University of Copenhagen and Department of Orthopedic Surgery, University of California San Diego. Lonnie was one of the first experts we talked with and she help us understand the general health issues that arise during space travel. Lonnie said that production of antibodies would be useful for space travel.

Lynn Rotschield, PhD, Senior Research Scientist, Bio and Bio-Inspired Technologies, Research and Technology Lead for NASA HQ STMD. Lynn visited us in Copenhagen in the spring. We had a very interesting and inspiring meeting, which among other reasons let us in the direction of a space project.

Mette Galsgaard, PhD Fellow Department of Chemistry, The University of Copenhagen. Mette has taught us how to make liposomes and supported lipid bilayers. She also helped with designing our liposome experiment.

Michael Hecht, Professor, Department of Chemistry, Princeton University. We met Michael at the European Meetup in Munich where Michael gave a talk. Following that we were very happy to talk with him, and he gave us some very nice input regarding protein folding and production.

Nikos Hatzakis, Associate Professor, Department of Chemistry, The University of Copenhagen. Nikos helped us designing the liposome and supported lipid bilayer experiment and made it possible for us to carry out our experiments using confocal fluorescence microscopy by letting us use the microscopes in his laboratory.

Piers Millet, PhD, Director of Safety and Security at iGEM and co-chairs iGEM's Safety Committee. Piers helped us with filling out the safety forms and pointed us in the direction of Jacob Hofman-Bang who helped us by making a biosafety analysis of our project. Piers also sent our safety questions to the Australia Group (safety council).

Poul Erik Jensen, Professor, Head of Copenhagen Plant Science Center, The University of Copenhagen. Professor Poul Erik Jensen has earlier been supervisor for the iGEM team from University of Copenhagen. He therefore kindly provided us a letter of recommendation which was helpful for our funding applications.

Søren Schmidt-Rasmussen Nielsen, PhD Fellow, Department of Chemistry, The University of Copenhagen. Søren has taught us how to use the confocal fluorescence microscope. He also helped with the design the supported lipid bilayer experiment.

Tomas Laursen, PhD, Postdoc at Section for Plant Biochemistry, University of Copenhagen. Tomas helped with preparing the protocol for liposome preparation and supported lipid bilayers and lent us extrusion equipment.

Virginia Wotring, PhD, Adjunct Associate Professor, Center for Space Medicine and Department of Pharmacology and Chemical Biology, Baylor College of Medicine. We had a very interesting talk with Virginia Wotring on skype, where we discussed several aspects of our project. We also got to discuss which protein would be relevant to produce from a pharmacist's point of view. Virginia suggested that we could look into production of IgG-stan (type of IgG antibody which is used therapeutically), antibodies, immune modulators (cytokines and interleukins) and G-CSF.