<p>After immense amount of discussion about what our project should be on from asteroid mining to water purification, we always came back to the same problem: how to release these systems into the environment safely. It was at this point we decided to focus our project on making a suitable chassis for GM release into the environment that overcomes of the potential problems that arise from GM release currently and combat some of the social stigma behind GMOs. We quickly found a system with potential, Minicells, which are achromosomal and therefore limit the exposure of these cells to the environment as they will degraded over time in a safe manner. From here our iGEM journey begins.</p>

             <p>After extensive research and talking to a few experts we determined that minicells were not suitable for our project. We found out that minicells had to be separated from the parental cell line and the method describe in literature for this was series of centrifugation which were time consuming. Which is a problem as these cells have limited time they are useful for and we didn’t want to waste the active timeframe of these cells in filtering them.

Therefore we began to looked at other methods for filtration including: different sized filters; Fluorescent-activated cell sorting (FACs); Dielectrophoretic (DEP); Field flow fractionation (FFF) and other microfluidic system. However, one of our supervisors pointed out that these may be faster than the centrifugation but are still slow; difficult to implement or do not work for our system.

We also came across the problem with minicells in plasmid localisation. Since the minicells were a product of incomplete budding at the ends of the cells, there were minicells that formed which would not have the plasmids and therefore are not useful. We tried to make a system the would localise plasmids to the ends, but this turned out to be very difficult and hard to implement.

We thought these were huge limitation to the project as we wanted our chassis to be accepted as an industry standard and for this it would need to be optimised and efficient. We therefore went back to the drawing board.  

During our research we found out about Maxicells, which was a system developed to identify proteins encoded in the plasmid. Maxicells are very similar to minicells but are bigger (hence the maxi) and were easier to work with as they don’t have the filtration problems minicells do and they can be selectively induced at any timepoint. So we decided to make Maxicells the basis for our chassis.

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             <p>Jane Calvert works on sociology of life sciences and is currently working on creating living things and therefore we thought she would be great to talk to as she has experience with the boundary between life and death. Since Maxicells have had their chromosome destroyed they are technically dead; yet they remain functional afterwards so are they still alive and how do we define death, especially in bacteria? During our meeting we discusses many different things from the boarder of cellular life and death and their definitions to what GMOs are. During our meeting some important questions came up such as the metabolism. Since our cells still produce proteins and function in this way this could be thought as life in one way. Another important question that arose from this line of though was how long can our Maxicell remain metabolically active for. This led us to the idea of measuring ATP levels and synthesis of fluorescent proteins over time, this ended up being an integral part of our project as this allowed us to quantify the timeframe our cells would remain useful for. We also discuss the problem with GM releases currently and Jane told us to look further into the laws around GM release and why they are so strict. </p>

             <h3 class=" text-light">Interaction with GM Inspectorates’ Office</h3>

             <p>After talking to Jane, we realised we should look into GM laws in the UK as our project aims to make GM release safer, it would be useful to know what the current regulations over GMOs are. Therefore, after some research we found out the governing body over GM laws in the UK is the GM Inspectorate and we go into contact with them to discuss our project. They told us our project sounded interesting and explained us about the process of getting approved for GM release into the environment. This was very interesting for us as we had no idea the about amount time and immense effort that goes into making a project suitable for GM release. After realising this we were more determined to make our project a success as we think GMOs are going to be a key tool in the future, that can make lives of millions better. However, without a suitable chassis this becomes difficult. Therefore, the idea of a safe chassis that follows these tight regulations over GMOs becomes even more desirable.  

             <p>After Researching Maxicells we knew we needed a specific mutant strain of E. coli (RecA¯ and UvrA¯) for the UV method of creating Maxicells to work efficiently. Therefore, we started by emailing around to see if another lab, at the university, had the strain we needed. Elise very kindly responded to our email and said to go visit her to as she had a large catalogue of strains. After visiting her we could not find the strain we needed. However, after explaining our project to her and our need to create achromosomal E. coli, she took us to speak with David Leach, the Head of School of Biological Science at the university. Professor Leach explained to us how his lab had developed two strains (DL2524 and DL3355) which can degrade DNA in an inducible manner and therefore create achromosomal E. coli easier than the UV method. He very kindly gave us the strains to use and after some testing we quickly realised the DL2524 strain had great potential for our project as it was healthier and easier to induce into Maxicells than the UV strain.  </p>

We had the idea of recoding the genome of our chassis to make it safer and prevent horizontal gene transfer but were having problem finding an easy to implement system for this. However, after presenting our idea for recoding the genome to St. Andrews on of their supervisors, Ariel , told us of a method for recoding the genes using an amber suppression mutation. He explained how a serine codon can be replaced with an amber stop codon and given our cell contains the right tRNA only our cell should be able to read the gene. He then referenced us to some papers which have done similar experiments. These turned out to be very useful and allowed us to create our whole semantic containment system.