Team:Dundee/Description

Dundee 2016

Project Description

BactiFeed

A novel solution to antimicrobial resistance?

Project Description

We are the undergraduate iGEM team from the University of Dundee and we’re Fighting Bacterial Infections.

Our project has developed from a team agreement in which we decided to work on antimicrobial resistance. The issue of antimicrobial resistance was identified as a growing concern, a “slow moving tsunami” (Dr Margaret Chan, WHO Director-General). We wanted to accept a challenging project which was achievable and would deliver a positive impact on a global crisis. The Review on Antimicrobial Resistance, commissioned by the UK Government and chaired by Lord Jim O’Neill predicted that by 2050 antimicrobial resistance will become one of the biggest killers exceeding cancer (Fig 1). It was also found that there was poor surveillance and data collection in most countries, however it is clear that the use of antibiotics in agriculture is widespread on a scale at least equivalent to use in humans, and it is projected to increase. Van Beockel et al. (2015) estimate that usage of antibiotics in agriculture will increase to 67% globally by 2030, and to 99% in Brazil, Russia, India, China and South Africa. Further, more than 70% of antibiotics deemed medically important for human health by the FDA sold in the United States are used in livestock, with most other countries above 50%. Clearly, the use of antibiotics has escalated to unsustainable levels and an alternative must be found.

Figure 1. Deaths attributable to AMR every year. Adapted from (1)

The Review also highlighted ten areas that they believed could have a huge impact on reducing the threat of antimicrobial resistance (Fig 2). Our team chose to research more about the effects of antibiotics in agriculture and the environment. We found that antimicrobial resistance in livestock also poses a Food Security issue. The quantity of antibiotics used in livestock is vast. In the US, for example, of the antibiotics defined as medically important for humans by the US Food and Drug Administration (FDA), over 70 percent (by weight) are sold for use in animals. Too many antibiotics that are now last-line drugs for humans are being used in agriculture.

Figure 2. Tackling antimicrobial resistance on ten fronts. We focused our attention on reducing antibiotics in agriculture and the environment. Adapted from (1)

Project Description

Antibiotic resistance occurs when bacteria develop mutations that reduce or eliminate the effectiveness of drugs, chemicals, or other agents designed to cure or prevent infections. The bacteria survive and continue to multiply causing more harm. By continuously using more antibiotics, we kill off any competition for these resistant strains and provide them with more resources to multiply. A bacterial strain can then pass resistant genes to a non-resistant strain, creating more resistant strains that thrive without competition. Clearly, a new method for removing bacterial infections is required.

Our team is displaying a method to combat specific bacterial infections without antibiotics, using proteins called bacteriocins that are secreted naturally by bacteria to kill similar strains in order to reduce competition (Fig 3). . We are engineering non-pathogenic E. coli to produce bacteriocins that will kill pathogenic strains of E. coli and similar bacteria such as Salmonella - these are just two examples of how our method works.

Figure 3. Schematic illustrates the competition between a target bacteria and an attacker cell. The attacker delivers a toxic effect through the release of bacteriocins and the uptake of these into the target cell. Bacteriocins contain three domains: translocation domain (orange), receptor binding domain (yellow) and a cytotoxic domain (blue). 1. Synthesis of bacteriocin and binding to its cognate immunity protein for protection of the host cell. 2. Release of the bacteriocin either through secretion or cell lysis. 3. Binding of bacteriocin to specific receptors on the target cells, and translocation of the toxic domain into the correct compartment for toxicity. 4. Toxic effect delivered resulting in death of target bacteria.

We believe our GM bacteria can be used in an animal feed, a ‘BactiFeed’, to remove common bacterial infections in livestock. Post-Weaning Diarrhoea in pigs is commonly caused by E. coli in the gut, and leads to dehydration, loss of body weight and death of infected pigs. To prevent mass infections and loss of income, farmers will administer broad range antibiotics to the whole herd after one confirmed diagnosis, resulting in a massive overuse of the antibiotics. We believe our BactiFeed will act as both a preventative measure for pigs that are not infected, but also a treatment for pigs that have already contracted the infection. Due to the enormity of these consequences, we consulted a range of professionals to discuss their thoughts on our project.

To achieve our goal, we can pinpoint the most specific bacteriocins for each of our target bacteria. These bacteriocins will then be designed to be produced by pH sensitive promoters and bile salt responsive promoters will be used to ensure the bacteriocins are released. Upon ingestion of our BactiFeed by livestock, the conditions within their gastro-intestinal tract stimulate bacteriocin production in the stomach and lysis of cells in the intestine, thus releasing the bacteriocins. To aid this, we can use mathematical modelling to determine the concentration of bacteria required per serving of BactiFeed to completely remove the bacterial infections in the chickens.

Aims of F.B.I: Fighting Bacterial Infections - BACTIFEED is all you need!

1. To use synthetic biology to address the emerging problem of antibiotic resistance.

2. To use synthetic biology to improve the characterisation of a number of promoters including two pH sensitive promoters (BBa_K1231000 and BBa_K1231001) and a bile salt sensitive promoter (BBa_K318514). We will clone gfp (BBa_E0840) downstream of these promoters to create the following composite parts (BBa_K1962014, BBa_K1962013, BBa_K1962010) in order to test the expression in conditions similar to that in the stomach and intestines of livestock.

3. To use synthetic biology to create a number of modularised colicins which lack a cytotoxic domain (BBa_K1962002 and BBa_K1962006) in order to develop novel toxins with specific warheads (BBa_K1962003, BBa_K1962007, BBa_K1962008) to target pathogenic bacteria. We will then test the heterologous expression of these toxins and their toxicity on target bacteria.

4. Mathematical modelling was carried out to aid in the understanding of our system and lead to further improvements.

5. To build an easy to use piece of hardware, which we can use for our proof of concept experiments. The piece of hardware should be designed to exert mechanical stress on our samples in order to simulate a stomach environment. It should also be designed in an environmentally friendly manner with as many recycled materials as possible.

6. Through every step of the way we have engaged with experts and the public to discuss our ideas and ensure that our product is tackling an unmet need.

References

(1) AMR Review 2016. https://amr-review.org/sites/default/files/160525_Final%20paper_with%20cover.pdf