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: the chassis that would be used is not safe for environmental release. It was at this point we decided to focus our project on developing a chassis that would overcome this problem. We quickly found a chassis with potential: Minicells - achromosomal E. coli cells formed my displacement of the FtsZ ring. From here our iGEM journey begins.
Minicells to Maxicells
After extensive research and talking to a experts in the field we found that minicells were not suitable for our project. Methods described in literature for purification of a minicell culture were lengthy and laborious iterative centrifugation. We began to look 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. Minicells also presented a problem with plasmid localisation. It could not be guaranteed that a plasmid would be taken up into the minicell with 100 % efficiency. 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 'easy use' 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 a plasmid. Maxicells are very similar to minicells but are bigger (hence 'maxi') and were easier to work with as they do not have the filtration problems that minicells did and they can be selectively induced at any timepoint. From here, maxicells became the basis for our chassis
Meeting Dr Jane Calvert
Jane Calvert works on the Sociology of Life Sciences, 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 metabolically active so are they still alive? How do we define alive vs non-living? During our meeting we discussed many different things from the border of cellular life and death and definitions as to what GMOs are. Another important question that arose from this line of thought was how long can our maxicell remain metabolically active for. This led us to the idea of measuring the active metabolic timeframe of our maxicells. Jane also advised us on the law and legislature around GMO regulation.
Correspondence with GM Inspectorates’ Office
After talking to Jane, we realised that 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 that the governing body over GM laws in the UK is the GM Inspectorate and we got in contact with them to discuss our project. They expressed interest in our project and outlined the process of getting approved for GM release into the environment. This was incredibly insightful as we had no idea of 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 bring improvements to the lives of many. 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.
Meeting with Professor David Leach and Dr. Elise Darmon
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 suggested we visit her 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.
St Andrew Meet up and Idea for Semantic Containment
After some correspondence with St. Andrews’ iGEM team, they invited us to a meet up with their team to discuss our projects and potential collaborations. It was a fun experience in St. Andrews where we met their and team and supervisors; presented our projects and most importantly got ice-cream. While we previously had the idea of recoding the genome of our chassis to make it safer and prevent horizontal gene transfer, we 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 suggested 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.
UK iGEM Meet up
This was a fun meet up between all the iGEM teams in the UK where we got to present our projects to each other; learnt about human practises and talked to other teams for potential collaborations.
Albert Bartlett and Modelling
From early in the project after discussions with experts such as Jane Calvert, we realised the main hurdle facing GM release currently is public and political perception. At a later point we reached out to Albert Bartlett, an agriculture company focusing on potatoes, on their opinions on GM release on their farms; William Jackson a representative from the company said this: “GM technology [such as your project] has the ability to dramatically change agriculture.” However, he also warned against the adoption of such GM technologies saying; “Until there is public acceptance of GM in EU, we will have no involvement in GMO technology”. In here lies the fundamental problem with GM release: Public Perception and acceptance. This lead us to the idea of modelling the absolute safety of our project by determining the failure rates of our semantic containment. Our model predicted that you were more likely to be struck by lightning, TWICE; in two consecutive years than for a read through to occur in a semantically contained gene. We hope to use these unbelievable stats to convince people of the safety of our project and the advances we have made to make a safe product. If people understand this and the great potential uses of GM a shift in perception is possible.
Talk with Synthetic Biology Consultant
As we want our project to be accepted in industry as a standard we wished to gauge opinions of experts in the field. For this we talked to an Industrial and synthetic biology consultant: Dr, Jon Dempsy. He told us our project sounded interesting and promising. He described to us how our system would be advantageous over a similar system with Chinese Hamster Ovary (C.H.O) cells which are often used in therapeutics and production of therapeutic molecules. He further explained that C.H.O cells are useful but produce a lot of unnecessary molecules using up valuable resources. Since our system is much more simple it would only produce our protein and therefore would be more desirable to industry. This gave us hope that our project would be one day accepted as a standard in industry and will make the use of GM much more efficient and safe.
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