This year's iGEM Copenhagen team is tackling a very interesting topic: production of proteins in space and on Mars! The motivation behind the idea is to provide the future Martians or astronauts with means of protein therapeutics production onboard the spaceship with minimal resources and risks of contamination.

Astronauts usually take with themselves painkillers, drugs fighting nausea or sleep promoting drugs and they are usually sufficient to attend the needs of a relatively short space missions. But once humanity decides to embark upon longer space missions and/or decide to colonize Mars the production of medicine in situ will become very attractive alternative to bringing medicine from Earth. The problem is that it is impossible to predict illnesses and thus decide which medicine to take with themselves and in what quantities. Another argument speaking in favour of protein production is a relatively short shelf-life of medicine, especially the protein ones.

This special class of drugs is protein therapeutics. They have few quite different characteristics in comparison to "normal" traditional drugs i.e. those we usually find in tablets. Besides inherited chemical instability, proneness to agglomeration and very specific pharmacokinetics there is also extreme selectivity, potency and low toxicity in comparison to "traditional" small molecule drugs. In certain cases they are the only available treatment for serious illnesses that are otherwise incurable (for example Non-Hodgkin lymphoma for which we use monoclonal antibodies such as Rituximab.

Current methods of protein production require extensive purification steps in order to acquire medical grade protein solution, and produce a lot of biohazard material, which together with high water/chemicals consumption makes it impossible to do onboard a space ship.

No protein therapeutics have been sent to space for human use yet… there hasn't been much need of it yet, but we can expect that once humanity embarks upon longer space missions that may very well change.

The idea

The project aims to use E. coli with type III secretion system (T3SS) cultured in a customised device as a simple protein production and purification system (Figure 1). The proposed device consists of two compartments. The first compartment contains the transformed E. coli with the gene of interest and the T3SS vector ( Figure 2). The tip protein of the T3SS complex will specifically recognises the biomimetic membrane and secrete the protein of interest through such membrane into the second compartment, the collecting chamber. The secreted protein is then collected in the buffer, which is free of contaminants.

Figure 1. Schematic representation of the proposed simple protein production and purification device (not to scale)

Figure 2. E. coli containing the T3SS vector and the expression vector on a membrane with porous support (not to scale)

The Membrane

The bacterial ability to induce the production of the T3SS is dependent on the recognition and interaction of the mammalian membrane. The specific requirements of said membrane needed to induce T3SS are still mostly speculative, though cholesterol has been shown to be essential to ensure effective T3SS formation and insertion into the membrane https://www.ncbi.nlm.nih.gov/pubmed/16800636

https://www.ncbi.nlm.nih.gov/pubmed/21182592

The ability of the enterohemorrhagic E. coli T3SS expressed in the E.Coli K-12 chassis to recognize artificial membranes is still largely unexplored, which necessitate proof of the bacteria's ability to recognize and produce the T3SS in response to our membranes. To this end, we will experiment using artificial biomimetic (cholesterol) supported membranes, liposomes and giant liposomes. After bacterial binding have been proven, unsupported membranes like artificial black lipid membranes or polymer membranes will be used to show protein secretion over the said membranes.

The Device

The chamber will allow testing the idea with minimal possibility of false positive result or contamination. It consists of 2 main parts; the lower collection chamber which will be filled with appropriate buffer (depending on the protein, most likely physiological solution or phosphate buffer), and the upper part, which will simultaneously hold the membrane in place with a silicone seal and allow for a certain volume of the medium with bacteria to be effectively stirred with possibility of later adding a flow system. A lot of effort has to be put into action to allow the sealing of the membrane to be tight, thus preventing bacteria to find its way to collection chamber (thus contaminating it) while also taking care of not breaking or /damaging the membrane. A special system of screws allowing for adjustment on force applied on the silicone ring has been designed for that purpose and is currently being tested on a pilot model.

There is of course room for further improvement of design. A flow system can be further developed allowing for the replenishment of nutrients and appropriate saturation of the media with gases/nutrients or bacteria. The lower collection chamber will have a rubber stopper (similar as on vials) allowing for small samples to be taken with syringe without disassembling the whole unit thus allowing for real-time-control of purity and /quality of the protein. Another idea would be to have a flow on the lower part connected with a UV-VIS spectrometer thus providing real time feedback about concentration of produced protein. Similarly, the upper parts of the chamber can be further improved. The membrane holder can be adjustable thus allowing for testing of different membranes/designs, while a flow system in the upper chamber will allow for the change of medium if needed. Implementing the modular approach will result in having the chamber made of 3 parts, each interchangeable, e.g. the lower collection chamber - different sizes and/or inlets, outlets, rubber stopper, transparent wall - the middle membrane holder - each designed for the specific membrane being tested - and upper media reservoir - allowing for different conditions to be maintained, i.e. double wall would allow for a water layer to circulate around a reservoir thus keeping the temperature constant, a magnetic mixer will be implemented allowing for more homogeneous conditions to be maintained and installation of inlets will provide means of controlling the media/bacteria concentration or nutrients.

Experimental Design

As previously mentioned, the goal of our experiments can be split into two:

1. To show recognition and attachment of SIEC to the different membrane types

2. To show secretion of target proteins over the different membrane types using the T3SS

Liposomes, supported lipid bilayers, egg yolk membranes and tobacco protoplasts are explored as different membrane types to test and characterize the T3SS for protein production.

Liposomes: Small and 'giant' artificial liposomes will be used to illustrate SIEC's ability to attach to an artificial membrane. The goal is to illuminate the membrane requirements for T3SS production.

Supported lipid bilayers: The supported artificial membrane will be used to SIEC's ability to attach to an artificial membrane.

Egg yolk membrane: Egg yolk membrane is easily obtainable and contains cholesterol. We wish to prove the ability of SIEC to both attach and secrete target proteins through a planar membrane using our own chamber design.

Values, human practices and outreach

The competition we are participating in is held by a non-profit organization called iGEM - international Genetically Engineered Machine. The purpose of the competition is to create solutions and generate knowledge of synthetic biology, which will be available in a big open source archive. Each team will provide at least one functional genetic construct (BioBrick) to the archive or improve an existing one. iGEM values sustainability in both economic, social, environmental and ethical aspects, and encourages the teams to not only make a novel project and improve or create a BioBrick, but also investigate how the final product might have an impact where it is implemented, and what problems it might meet or cause. The investigation is in iGEM terms called human practices, and is meant to make the teams more conscious about the choices made concerning the idea and product development process as well as the design and marketing. iGEM also emphasizes public awareness and knowledge of synthetic biology and therefore encourages teams to communicate their project to the local media as well as national or international ones in order to raise awareness and facilitate dialogue.

Our human practices investigation

In the idea development phase, we have carefully discussed different uses for our protein purification system and further discussed how it will impact society and the environment. Since our project will allow for simpler protein production, this would be useful in places with little to no infrastructure such as the third world or even Mars, or it could be useful in smaller companies or new biotechnology startups. Another question also arises; will the easy access to protein production also allow for easier access to illegal or dangerous substances or bioweapons.

Although not a weapon in and of itself, the T3SS is an example of a part that could prove dangerous. Biosafety is, therefore, a high priority to us, and we want to research the risks and strive to make our end product as safe as possible. Investigating safety, we are hoping to cover related topics such as which laws deals with GMO bacteria on Earth as well as which laws function in space, where no single country can claim ownership. We plan to collaborate with experts in biosafety and representatives of our future user base to create an end-product that's sustainable and useful to the industry and society at large.

Chemical waste, carbon footprint, the spreading of antibiotic resistance and GMO release are all topics that any sustainable organization should be conscious about. We have chosen a project that is going to be limited to a controlled laboratory setting, where people are educated in the handling and proper elimination of unsafe organisms and substances. Our method removes the need for several purification steps from the the standard process of bacterial protein production. This removes the need for a lot of water and chemicals, which the standard method requires during these steps, as well as sparing valuable time for the user. We aim towards making our methods a greener and more sustainable way of producing proteins in the future using bacterial hosts.

Our outreach and PR

We're currently present on Facebook, Instagram and Twitter. While our Facebook and Instagram profiles are meant to be funny, casual and explain our project to friends, family and other students at the science department, we're striving towards a more serious voice on Twitter, in order to reach the scientific community and all the international iGEM teams. We also have a blog on Medium, where we're writing about the project and the science behind it in a light and informative voice. We're imagining that this blog will be interesting for other iGEM teams, and help recruit science students for future iGEM teams.

We are working on getting media coverage, in order to raise public awareness on synthetic biology. In our meeting with the public, we are naturally very aware of the brands that have chosen to support us. We will either mention them, wear the logos or both according to agreements.

Design of chamber

Introduction - what is Bacta_tank3.0?

The final product of PharMARSy, Bacta_tank3.0 would be a device that allows for growth of bacteria in one chamber and secretion of pure protein into another. The chambers would be divided by a biomimetic membrane, which serves two purposes: it should allow for bacteria to secrete the protein through membrane into collection chamber using injectosome - a syringe-like protein structure, but at the same time it should be robust enough to prevent bacteria to crossing into lower chamber thus contaminating the protein solution.

The design of the chamber should therefore fulfill the following criteria:

Simplicity of handling: easy to open, easy to safely close
Allow for fixation of membrane without breaking it (sufficient control over applied pressure)
Prevent mixing of media from different chambers
Allow for fluidic system / stirring / sample taking

Due to popularity of 3D printing and its relative accessibility we decided to print our own prototype. But it had to be designed first...

Design of the Bacta_tank

The first design of the chamber was made before deciding on what type of membrane to use. It was therefore crucial to keep the design open for changes and later modifications.

A general concept was disk-like plastic container allowing for delicate handling of the presumably fragile membrane (fig 1).

Figure 1: First design.

It was realised that the membrane will have to be somehow held in place, to prevent leakage and to allow for stirring of the media above it. The design was therefore improved to address that problem. One solution was to include another part that would hold the membrane in place as can be seen in figure 2 and 3.

Figure 2: Multicomponent design - Bacta_tank1 offers more yet at the cost of simplicity. It served as a template for further development, but was never printed.

Figure 3: Bacta_tank2, middle part.

Figure 4: Bacta_tank2, upper part.

However all of the models described above are relatively complicated for handling, and not that easy to manufacture with 3D printers techniques available to us. If possible, we were aiming at designing a prototype that would be easy to produce and easy to handle.

A radically new design was needed - simpler and smaller.

The inspiration came from the very thing that we were trying to get rid of: the screws. Why having external, metal screws if one can design chamber components to be "big hollow screws?"

Inclusion threads inside the walls of chamber components meant that parts can be simply screwed one into another, eliminating any need of external holders or screws. Admittingly somewhat a challenge for a designer and a test of precision for 3D printer, at least it would be very simple for the end-user.

It is relatively simple to design a 2 components system where only 2 threads are used, however when having thread on both sides of the middle part (coloured blue in Figure 6), one has to pay attention not to "slice" with the threads through the walls of the body of the component. To make screwing easier, a hexagon extension was envisioned at the top of two upper parts allowing for use of tools if necessary.

An example of cross section can be seen in the following illustration (Figure 5).

Figure 5: A cross section of design for Bacta_tank3. Components can be seen to fit nicely and a simulation was performed many times to check for any issues prior to printing.

Figure 6: Another view of the Bacta_tank3 design, both assembled and disassembled.

As can be seen on the picture, including threads considerably simplified the use of "Bacta tank". The membrane would be placed between the upper and middle part, and protein wouldl be collected in the lowest chamber, while bacteria culture would be cultured in the utmost top. The threads allow for customisation of the volume of collection buffer in collection chamber, while also allowing for careful fixation of the membrane between upper two parts.

Figure 7: Rendering tool in Autodesk Fusion 360 program allows for different materials to be "tested" for visual appearance prior to actual production. Of course printing in glass or aluminium was beyond our capabilities.

Technical details

The drawing of the chamber design was done in Fusion 360, a cloud-based CAD/CAM/CAE tool for collaborative product development, using an educational licence. The version used was 2.0.4126:

The drawing was then exported into Ultimaker Cura, an open access program for preparing the model for 3D printing.

All chamber components were printed Ultimaker 2+, using PLA filament (polylactic acid), with filament diameter of 2.85mm and Marine Blue color, with disabled support, enabled print cooling and adhesion plate type "Brim".

Future improvements

Of course PLA polylactic acid is probably not the best material for growing bacteria in. At least some painting could be used after printing the model.

The device that was envisioned for astronauts to take on their space voyages would be of course far more advanced. It should be hermetically closed with double walls and multiple inlets/outlets allowing for manipulation of liquids and monitoring of parameters. It should also be enclosed in one more contamination containment chamber (the principle similar to what they currently use in nuclear power plants) which would protect the crew from the spillage of biohazard material in case of accidents. The fluidic system should allow for uninterrupted sampling both of the bacteria culture and of the protein solution. Bacteria could mutate due to exposure to mutagenic cosmic radiation so checking the genome of bacteria prior to start of protein production could be a good idea. On the other hand a samples from protein solution could be analysed either via capillary electrophoresis on a microfluidic device or some other method that would confirm the required purity and "quality" of the protein prior to use on the patient.

Finally, the chamber must withstand the disinfection between changing the bacterial strains when different proteins are needed. The most vulnerable part in this case would probably be the membrane, so a system that allows for changing of the membrane is a much better solution than the system with fixed one. This would also considerably extend the useability of the system, since a membrane that wears out could be easily changed.

Pictures from real process:

A special note to the 3D printing enthusiasts. As the principal idea of 3D printing is accessibility to everyone (who has access to the 3D printer) and sharing, many designs can be found online. This is the case also with our design. If you are interested into our Bacta_tank3.0 and would like to print it on your own, please feel free to contact us and we will readily share our knowledge with you. We are also welcoming any suggestions from the more experienced printer-maestros, since we are very much aware that there is plenty of room for improvement.

Figure 8: Ultimaker 2+ that was used for printing. The blue duct tape was used due to problems with sticking to the baseplate.

Figure 9: The initial layer is usually the hardest. A blue adhesive tape tends to solve the problem, especially if combined with the hair conditioner sprayed just before printing.

Figure 10: After first layer is printed, the worst is over and the rest of the body can be printed without supervision.

Figure 11: Top view

Figure 12: Right after finished printing, a careful removal of the object follows.

Team:UCopenhagen/Description

Project Description

Introduction