Difference between revisions of "Team:UCopenhagen/Description"
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Revision as of 11:09, 14 October 2018
Project Description
Introduction
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.