Team:CLS CLSG UK/Poster

Poster: CLS_CLSG_UK

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Project CocEels: a genetic engineering solution to cocaine in the Thames

Team

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.

Introduction

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.

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 in order to breakdown 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. So 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 a hypoxic 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 due to the up regulation of MazF and down regulation of MazE, 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.

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. In addition, we realised that there are many other drugs (apart from cocaine) which cause great harm to the aquatic ecosystem. One of the most important of these would be antibiotics. 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. Aptasensors are biosensors which use aptamers (short DNA strands) as the biological target recognition element. As current detection methods are heavy, large and extremely expensive, we designed a cheap and effective electrochemical aptasensor, as seen in the diagram below.

The Aptamers would be immobilised upon the gold working electrode. These aptamers would change shape in the presence of target molecules, hance causing a change in current. This change in current would be detected by the potentiostat, an analytical tool used in many aptasensors. The aptamers for cocaine and antibiotics worked slightly differently, as seen in the diagrams below.

Cocaine Aptamer

Antibiotic 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. These potentiostats are usually very expensive (often ranging into a price range of above £5000). We planned on using a chip sized potentiostat designed by Orlando Hoilett and his team at Purdue university, and modifying it to allow for all programs to be run through only an SD card. Not only is our potentiostat extremely small and portable, but also extremely cheap compared to other potentiostats, costing only around £150. Please see our circuit diagram below. You can access our github here (insert link) for the arduino code.

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.

Results

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.

References and Acknowledgements

If not already cited in other sections of your poster, what literature sources did you reference on this poster? Who helped or advised you?