Team:CLS CLSG UK/Design

Project Design

Project Design

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Design

We wanted to address the issue of cocaine entering the rivers of Europe, specifically the River Thames. In order to do this, the first thing we had to research was the best way by which to break down cocaine. We wanted to use a biological method and our research first presented us with the Cocaine esterase enzyme from humans. This enzyme seemed perfectly suitable, except for the fact that in the presence of ethanol the cocaine is not metabolised normally but instead converted into cocaethylene, a far more potent and toxic form (this is often one of the complications with people who co-abuse cocaine and alcohol).[1] After doing some further reading onto cocaine toxicity in humans we found that one of the most promising treatments is the use of a bacterial cocaine esterase and so we landed on the cocaine esterase from Rhodococcus.[2]

Rhodococcus lives in the rhizosphere of coca plants and so uses cocaine as its primary carbon source. It is able to metabolise cocaine into the same metabolites as the human enzyme, except it is also able to break down cocaethylene.[3]

We then looked at how best to produce our enzyme, originally we looked to incorporate this enzyme into a self regulating circuit with tetracycline. This would help to reduce the cell stress, but would reduce the amount of enzyme produced overall. However, after some further consideration, and some very preliminary modelling, we realised that maximising the rate at which we can break down the cocaine was the most important part of our system and so maximising the amount of enzyme produced was a priority. With this we had constructed our first genetic circuit: the constitutive production of the cocaine esterase enzyme.

After we ran our full model, we also realised that the low thermal half life of the wildtype enzyme was reducing the ability of our system to break down cocaine as efficiently as possible. After going back to the drawing board, we discovered that a mutant form of the enzyme, E172-173, has a significantly increased half life over the wildtype. This form is currently undergoing phase two trials as a cocaine overdose treatment due to its high catalysis rate as well as its improved thermal stability.[4] For these reasons, we have decided to use this enzyme in our system, due to its significant improvement over the wildtype enzyme in a whole host of ways.

After we decided on how we wanted to tackle the problem we had to find a location and method by which we could implement it. Given that all the cocaine that entered the Thames, and other rivers around Europe, entered through the sewer system, this seemed an apt place to start. When looking at the sewer system we had to find a suitable location and also find a way of containing or immobilising our bacteria. After some in-depth, research we constructed this basic breakdown of the sewer system:

Our main issue with all the parts of the sewer system was the speed at which the sewage passes through most of the tanks; at these speeds our bacteria would not have enough time to break down the cocaine. Therefore, we chose the primary settling tanks as the location of our modified bacteria. In these tanks the flow is slowed to allow for solid particulates to settle to the bottom and for the liquid at the top to be siphoned off for the rest of treatment. These tanks are the ideal location to break down cocaine due to the very slow flow rates, giving our E. coli time to degrade substantial amounts of the cocaine. During research, we also realised that much of the reason why drugs and other harmful chemicals are easily flushed into rivers is due to overflow of sewers systems during storm surges. We realised that the secondary sedimentation tank, along with another host of treatment processes, are bypassed when inflow of water is too high, leading to the spikes in pollutant concentrations. However, the primary sedimentation tank is not bypassed, and so we planned on placing our bacteria in both primary and secondary settling tanks.

Within these tanks we then had to look at the way in which we could immobilise the E. coli. Many past iGEM teams use biofilms, and while we looked into this, we could not get over the very high levels of antibiotic resistance. In European rivers, especially the Thames antibiotic resistance is already such a large problem, so we didn’t want to solve one problem while contributing to another. In addition, we realised that such biofilms could potentially block the primary sedimentation tank, causing further disruption to sewer systems. We also looked into other immobilisation techniques, incuding a molecular tea bag made out of poly tetrafluoroethylene membranes and lining the entire tank with agarose gel. All of these proposals had significant issues with either the cost or the practicality, so we continued to search for more innovative solutions. Soon, we discovered a paper titled: ‘Programming Controlled Adhesion of E. coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins’. What this paper lined out was a novel genetic construct that is expressed on the outer surface of the membrane which allows for adherence to a complimentary antigenic surface.[5] It was exactly what we had been looking for.

The extracellular protein worked by combining the very stable beta barrel from intimitin, which is found in enterohemorrhagic and enteropathogenic E. coli strains, with an immunoglobulin domain (here shown as a VHH). The VHH has a complementary shape to an antigen that can be bound to a plastic surface.

There is a large library of VHH domains as well as their complementary binding proteins. In this way, the synthetic adhesin (SA) can be adapted for many uses and many situations. In our case we wanted our antigenic protein to be GFP, due to its low risk, high availability and high familiarity amongst iGEM teams. In this way, by binding GFP to a plastic surface the SA on the membrane of the E. Coli is then able to bind to that GFP and in itself be bound and immobilised on the plastic surface. The results of the experiments using the SA were very promising as published in the paper. However, we wanted to see for ourselves and our results were very much in line with their findings. We are the first iGEM team to propose a method like this for the immobilisation and adherence of bacterial cells. Our results show great promise for other teams in the future to use this construct in order to further their own projects.

Source: Reference 5

Within the settling tanks we needed a surface to bind our E. Coli . The inside of the tank, while the obvious option, seemed to be impractical and also have a low surface area to volume ratio. However, within many settling tanks are additional structures known as lamella clarifier plates. These are angled plates with very high surface area to volume ratios, designed to slow down the flow within the tanks (please see diagram below showing the position of lamellas in the sedimentation tank). After analysing a number of different shapes for lamella clarifiers, we decided to use a chevron shaped design for the following reasons [6]:

  • It provided the highest surface area to volume ratio amongst all the other plates available
  • Its 60 degrees angle of elevation provides greatest decrease in flow rate
  • Unlike circular and hexagonal designs, its unique v-shaped structure prevented any disproportionate accumulation of sludge, hence preventing any blockages
  • Comparatively small hydraulic radius (at 25mm) allows for greater surface area for bacterial binding, hence allowing larger amounts of cocaine to be broken down
Source: Hewi plastics technology

Now that we were placing our E. Coli into the sewer system it was brought to our attention that we would have issues if genetically modified bacteria were able to escape into the river and the wider ecosystem. We needed a method by which we could endure their containment. Looking at previous iGEM teams we realised that we did not need to necessarily contain them if we could ensure cell death upon their escape. So we began looking for parameters which we could use to design our kill switch. We first looked at light, since the sewer system is often dark and the ecosystem is clearly outdoors. But the uncertainty regarding whether all of the water treatment system is in darkness, as well as that fact that the bottom of rivers is very dark, meant that the success rate of our containment method, using light as the trigger, would be low. We continued looking and realised that we could use specific processes in the sewer itself as the trigger for our kill switch. By this, we meant that further along during sewage treatment, both anoxic and hypoxic conditions are created and we wanted to use this change in the environment to begin the apoptotic cell death of our E. Coli, preventing them from affecting the ecosystem.

When looking at kill switches we realised that we needed to use a sensitive but dynamic system and so opted for the MazF-MazE toxin-antitoxin system. While this system has been used as a kill switch by previous iGEM teams it had never been done using hypoxic conditions as the trigger. Furthermore, the design of our kill switch is far more efficient than previous uses due to the independent control of the toxin and the antitoxin.

We created the kill switch using the relative strength of the CMV and hypoxia induced promoter which was done by team SCUT_China 2015, as well as the relative strength of the differing RBS which can be found in the literature. This means that in normoxic conditions the amount of MazE produced will be greater than the MazF produced and so the cell will remain alive…

Whereas in hypoxic conditions the amount of MazF produced is far greater than the level of MazE and so the cell dies due to the effect of the toxin...

Therefore, our system (i.e. the combination of all three circuits) allows for a new and innovative way of immobilising E. coli bacteria while allowing rapid breakdown of cocaine early in the wastewater treatment process. Moreover, our novel hypoxia induced MazF-MazE toxin-antitoxin kill switch has been designed to act as a safety net, almost eliminating any risk of bacterial spread.

References

  1. Dean, R. A., Christian, C. D., Sample, R. H. B., & Bosron, W. F. (1991). Human liver cocaine esterases: ethanol‐mediated formation of ethylcocaine. The FASEB Journal, 5(12), 2735–2739. https://doi.org/10.1096/fasebj.5.12.1916095
  2. 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
  3. Bresler, M. M., Rosser, S. J., Basran, A., & Bruce, N. C. (2000). Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase from Rhodococcus sp. Strain MB1. Applied and Environmental Microbiology, 66(3), 904–908. https://doi.org/10.1128/aem.66.3.904-908.2000
  4. 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
  5. 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
  6. PP structured fill block Hewitech. (2015). Http://Twitter.Com/DirectIndustry. https://www.directindustry.com/prod/hewitech/product-90421-2257686.html