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Revision as of 22:55, 10 November 2020

Poster: CLS_CLSG_UK



Please click on the various sections of the poster in order to see different parts of our project. If you are viewing this on the mobile version, please scroll to the bottom after clicking on the boxes in order to view text.

Student leaders: Yashaar Daad and Louis Gringras

Team members: Isaac Tolley, Isabella Yeo Frank, Lucza Lj, Jared Stoloff, Asad Khan, Ela Tabbouche, Milla Ivanova, Kaavya Kanagarajah, Rohan Prakash, Sahil Shah, Ethan Santos, Yeshin Yoon, Peter Heywood, Joseph Hee, Cassidy Ashworth, Jian Hui Mo, Lucas Czajka, Arabella Henderson, Alexander Wallop, Nathan Leung and Gautam Krishna.

Primary PI: Mr Simon Hall

Secondary PI: Dr William Tibbits

Abstract
As a school located on the banks of the River Thames in London, we are focussing on the impact of cocaine upon the critically endangered European Eel, Anguilla anguilla. Concentrations of cocaine as low as 30 ng/L have been shown by studies to have detrimental impacts upon their migration and breeding patterns. We aim to solve this problem by using genetically modified E. coli bacteria immobilised in the primary sedimentation tanks of the sewers. We also designed a novel, hypoxia induced toxin-antitoxin kill switch which will destroy our bacteria in the presence of standard anaerobic processes in wastewater treatment plants and hence prevent our bacteria from spreading into the wider ecosystem.

We are the CLS_CLSG_UK 2020 iGEM team!

Since 2016, our team has only consisted of members from the City of London School for Boys. This year is the first time we have a joint team, incorporating members from the City of London School for Girls as well and we are extremely excited to present our project.

When setting up our project, we created some basic goals:

Local

We wanted to create a project which would impact our local community. A project which would make us unique.

Innovative

We wanted to be new. We always wanted to have a sense of innovation and novelty with regards to our project. We wanted to use existing concepts and put a new spin onto it, while maintaining a cool simplicity to our idea.

Responsible

From the very onset of our project, we knew that we needed to create a project which was responsible for the environment and our wider society. A project which would solve problems instead of creating new ones.

After a lack of success in 2019, our 2020 team was dedicated to producing a unique and innovative project. We immediately wanted to address one of our core project goals: a local problem with a local solution. As a school located on the banks of the River Thames, at the heart of London, we believed our environment presented us with a key opportunity.

As our team members would walk home from school, many would often see fish, particularly eels, lying dead on the banks of the river. We researched this further, and realised the effect of drugs on the aquatic ecosystem in the Thames. Please see our ‘Problem’ section for further details.

The massive cocaine problem in London affects more than just the people, much of the large quantities consumed are excreted and so make their way into the rivers and wider ecosystems around the cities. The ramifications on wildlife have already been significant and if nothing is done to stop it, then the world could be looking at an extinction level event.

Cocaine usage in London compared to some other European cities

The largest effects of this pollution are felt by the Anguilla anguilla or the European Eel. It is currently critically endangered as its populations have seen the steepest downwards trend of any freshwater fish. The cocaine causes degradation of their skeletal muscles that will prevent them from migrating to their breeding ground in the Sargasso sea. This means there are less eels to mate, so less new eels being born each mating season, and so their population continues to decline.

As well as this, they face many other substantial issues, from hydroelectric power stations and damns to overfishing and global warming.

Our project aims to place genetically engineered bacteria into the primary sedimentation tanks of the sewers (these regions have relatively slow flow rates and so are good for immobilising bacteria).

More specifically, we plan on placing our bacteria on lamella clarifiers, plastic structures present within these tanks which have a very high surface area to volume ratio and are best suited for our purposes.

Source: Hewi plastics technology

Our bacteria would be immobilised upon these lamella clarifiers and modified to break down cocaine very efficiently. Please see our design page for further details.

When approaching this problem we needed to construct three different genetic circuits.

Cocaine Esterase Production

The first was the production of the cocaine esterase enzyme for breakdown of cocaine:

The cocaine esterase hydrolyses the ester bond in the cocaine, thus producing benzoate and ecgonine methyl ester. While the enzyme originated in Rhodococcus sp. our system would use a mutant form of the wildtype, with a significantly improved half life.

Synthetic Adhesin Expression

The DNA for the expression of the Synthetic Adhesin was integrated into the chromosome of the E. coli and through its expression on the outer surface of the membrane adherence to a plastic surface would be greatly improved:

The Synthetic Adhesin has an anchoring region, based off of the protein Intimin. It also has a complementary binding region that protrudes out from the membrane and can bind to any complementary protein. If complementary proteins are bound to a plastic surface, then the Synthetic Adhesin is able to bind to the surface and thus the E. coli can adhere.

Hypoxia-induced Kill Switch

The third circuit is a hypoxia induced kill switch that has been designed to prevent the modified E. coli escaping into the wider environment. To do this we utilise the fact that sewage treatment involves an anaerobic stage, and so in hypoxic conditions the our bacteria will not be able to survive:

While in normoxic conditions:

The kill switch utilises a the MazF-MazE toxin-antitoxin system. By placing the two proteins under the control of different proteins, we are able to ensure cell death in hypoxic conditions while sustaining growth in normoxic conditions. The implementation of the toxin-antitoxin system in this way solves the issues exhibited with previous uses and designs.

Our model aimed to calculate the rate at which the two enzymes would break down cocaine. To do this we created three separate models. The first to give us the rate of cocaine diffusion.

The next was to calculate the two different enzyme concentrations in the cell - the enzymes differed due to the increased half life of the mutant. To do this we created an iterative model for both enzyme concentrations in the cell.

The final model utilised the Michalis-Menten equation and allowed us to get a breakdown rate for both enzymes.

Then using data from the modelling we were able to obtain values for the total breakdown of our system in situ utilising the different enzymes. This gave us the value of breakdown of the wildtype as = 2.3 pgs-1. While the mutant breaks down cocaine at a rate = 1.1 mgs-1. Given the flow rate of cocaine = 0.000316 g s-1, this demonstrates that our model predicts that with the mutant enzyme our system would be able to break down the cocaine in the sewer in its entirety.

We realised that in order to monitor the efficiency of our circuits in the sewer, we would require a quantification mechanism capable of detecting drug concentrations in the effluent flow. We also understood that there are also many other drugs (such antibiotics) which cause great harm to the aquatic ecosystem. Large amounts of antibiotics in effluent flow can result in high bacterial resistance, having devastating impacts upon local ecosystems.

We developed a device capable of sensitively detecting cocaine and antibiotic concentrations. As current detection methods are heavy, large and extremely expensive, we designed a cheap and effective electrochemical aptasensor, using two different aptamers for detection of cocaine and tetracycline. Please see their mechanisms below.

Antibiotic Aptamer

Cocaine Aptamer

In order for our device to operate, we would also require a potentiostat to control the voltage into the electrodes and record readings from our device. Our potentiostat has a number of great advantages over other models:

  • It is comparatively cheap, costing only £150 whereas normal potentiostats start at around £5000
  • It is extremely small and portable, being the size of a small arduino chip
  • All programs can run through only an SD card, allowing even greater portability

Please see our circuit diagram below. You can access our github here

Our potentiostat also would give timed readings to a peristaltic pump, allowing sample to be replaced every 30 minutes. As a result, our device would allow automated, in-situ samples to be taken. Such a device would require very little human intervention, allowing readings to be taken even in potentially toxic environments.

As our project strives to solve an issue within the River Thames, we had to contact several organizations to fully understand the potential implications it could have, to ensure its effects were solely positive. We achieved this through contact with the Sustainable Eel Group, London Zoo and Thames Water, which was vital for us to comprehend the impacts from an industrial and environmental perspective.

It was surprising to find out that many small fish are damaged a lot more than the Eels by the chemicals, meaning our project would have a more widespread positive impact than we had originally expected it to. However, the Sustainable Eel Group highlighted that a key factor in the demise of the European Eel population is the man made additions to the River Thames, such as hydroelectric power plants. Following this information, our team decided that as well as creating our project we ought to raise our concerns about the European Eel population to those who can create legislation to protect the eels, so we wrote a letter to the EU.

Another important part of our project and our human practices was our science communication as we felt it was incredibly important to inspire interest in the future of biological technology. We made a short video for the lower school and wrote leaflets for students, parents and teachers at our schools. As well as this, we found it particularly important to teach girls about developments in science and stress the importance of having women working in STEM, so we held a session where we showed them the short video and encouraged them to email us, organise a meeting and learn more about iGEM and our project.

With an aim of improving our project while also learning more about the diverse applications of synthetic biology, the CLS_CLSG iGEM team has collaborated with multiple university and high school iGEM teams from around the world by exchanging emails and texts as well as having meetings on Zoom or Google Meets. The products of our collaborations include raising awareness about important issues faced by the world today, from both a medical and environmental standpoint.

We tested out synthetic adhesin using a protocol based around ELISA plates. The results of this testing gave us very convincing qualitative and quantitative data.

Our qualitative data consisted of images of the wells taken at 100x magnification.

This image shows the stark increase in adherence achieved through use of the synthetic adhesin.

Further to this we obtained some quantitative data with which we were able to run an independent students t-test.

The results of this statistical analysis shows that there is a statistically significant difference between the E. coli expressing the synthetic adhesin on the antigenic surface to an other E. coli in any conditions that we tested them in. This is true for a 98% certainty.

Problem

  1. Why the once common European eel is now Critically Endangered (and what can be done about it). (2019). Wwt.Org.Uk. https://www.wwt.org.uk/news/2019/06/14/why-the-once-common-european-eel-is-now-critically-endangered-and-what-can-be-done-about-it/17073#
  2. Verreycken, H., Belpaire, C., Van Thuyne, G., Breine, J., Buysse, D., Coeck, J., Mouton, A., Stevens, M., Van den Neucker, T., De Bruyn, L. and Maes, D. (2013) IUCN Red List of freshwater fishes and lampreys in Flanders (north Belgium). Fisheries Management and Ecology 21: 122–132.
  3. Munro, K., Martins, C. P. B., Loewenthal, M., Comber, S., Cowan, D. A., Pereira, L., & Barron, L. P. (2019). Evaluation of combined sewer overflow impacts on short-term pharmaceutical and illicit drug occurrence in a heavily urbanised tidal river catchment (London, UK). Science of The Total Environment, 657, 1099–1111. https://doi.org/10.1016/j.scitotenv.2018.12.108

Idea

  1. Tchobanoglous, G., Stensel, H., Tsuchihashi, R., Burton, F., & N/A Metcalf & Eddy, Inc. (2013). Wastewater Engineering: Treatment and Resource Recovery (5th ed., Vol. 1).
  2. PP structured fill block Hewitech. (2015). Http://Twitter.Com/DirectIndustry. https://www.directindustry.com/prod/hewitech/product-90421-2257686.html

Design

  1. Cooper, Z. D., Narasimhan, D., Sunahara, R. K., Mierzejewski, P., Jutkiewicz, E. M., Larsen, N. A., Wilson, I. A., Landry, D. W., & Woods, J. H. (2006). Rapid and Robust Protection against Cocaine-Induced Lethality in Rats by the Bacterial Cocaine Esterase. Molecular Pharmacology, 70(6), 1885–1891. https://doi.org/10.1124/mol.106.025999 Rational Design, Preparation, and Characterization of a Therapeutic Enzyme Mutant with Improved Stability and Function for Cocaine Detoxification. (2014). Acs.Org. https://pubs.acs.org/doi/10.1021/cb500257s
  2. Programming Controlled Adhesion of E. coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins. (2015). Acs.Org. https://pubs.acs.org/doi/abs/10.1021/sb500252a

Hardware

  1. Mehlhorn, A., Rahimi, P., & Joseph, Y. (2018). Aptamer-Based Biosensors for Antibiotic Detection: A Review. Biosensors, 8(2), 54. https://doi.org/10.3390/bios8020054
  2. Yang, Z., Ding, X., Guo, Q., Wang, Y., Lu, Z., Ou, H., Luo, Z., & Lou, X. (2017). Second generation of signaling-probe displacement electrochemical aptasensor for detection of picomolar ampicillin and sulfadimethoxine. Sensors and Actuators B: Chemical, 253, 1129–1136. https://doi.org/10.1016/j.snb.2017.07.119
  3. Hoilett, O. S., Walker, J. F., Balash, B. M., Jaras, N. J., Boppana, S., & Linnes, J. C. (2020). KickStat: A Coin-Sized Potentiostat for High-Resolution Electrochemical Analysis. Sensors, 20(8), 2407. https://doi.org/10.3390/s20082407

Acknowledgements