How BactiFeed Works
Bacteriocins modified to target specific bacteria
From Farm to Fork...
Food security within the agricultural environment is what we are targeting, in particular focusing on bacterial infections within livestock and drug resistance. Food security begins right at the beginning of the production line: at the farm. The industrialisation of animal production was made possible by the availability of antibiotics for livestock and poultry. The livestock must be kept healthy in order to reach food security standards, not only is this important to the agricultural environment but it also means a healthy economy for the farming industry. Antibiotics are currently used in feed to protect livestock from infections as well as to generally boost their growth, however drug resistance is causing large problems and the more antimicrobials are abused the worse the situation gets.
Figure 1. Misuse of antibiotics in livestock can have many downstream effects, including the increased potential of the emergence of resistant human pathogens. Fig from (1)
Discovery of new antibiotics is at an all-time low; it has been 30 years since a new class of antibiotics was last introduced. Only 3 of the 41 antibiotics in development have the potential to act against the majority of the most resistant bacteria.
Our mission is to use synthetic biology in order to solve this problem and to create an array of novel toxins by modularising the architecture of bacteriocins.
Figure 2. Timeline showing the date of discovery of antibiotics and the date of resistance. (2)
We will be focussing on a specific set of bacteriocins known as colicins. Colicins are bacteriocins produced by E. coli and are proteins produced and secreted by bacteria in order to eliminate closely related competitor strains. We aim to generate novel toxins by alternating the cytotoxic domains with different warheads. This gives us the ability to design selective toxic domains targeting only pathogenic strains. This approach has a great advantage over antibiotics, which are often rather non-selective. The livestock produce can now pass the food security standards without impacting global antimicrobial resistance and land safely on the consumer’s fork.
Figure 3. Colicin structure showing the three domains of a colicin. Possibility of interchanging the toxic domains (Warheads) at the multiple cloning site of bacteriocin structure allows for less chance of resistance and more specificity.
...And From Beak to Bum
Our idea was to create a feed for livestock (in this case we focused on chickens), which is coated in our synthetic colicin producing bacteria. This is BactiFeed. The concept is simple; the chickens eat BactiFeed, as the bacteria travel through the GI tract of the chicken they begin to produce these synthetic colicins. The colicins are not active when expressed in the host cell as the host expresses a corresponding immunity protein to the colicin. In order to release the colicins to allow them to target unwanted bacteria the producing cells must lyse.
Figure 4.Illustration of our proof of concept. Our bacteria producing our synthetic toxins will be expressed in the presence of low pH and then the cells will lyse and release the synthetic toxins in response to bile salts.
Starting from the beak lets follow the journey of our BactiFeed. After ingestion the bacteria reach the chickens stomach. In a chickens case there are two stomachs called the proventriculus and the gizzard. These have a low pH environment so we decided to begin our plasmid construct with a pH sensitive promoter to induce production of our synthetic colicins. Throughout the following 20 minutes (or less depending on the individual chickens digestion speed) the bacteria begin to synthesise colicins that build up within the cell. The next stop for our bacteria is the intestine, at which point they encounter bile salts produced by the chicken’s liver. Naturally the next part of our plasmid design included a bile salt induced promoter with a lysis cassette downstream of it. This lyses the cells releasing the colicins to travel to their targets and kill them. The journey has now come to an end and the chicken’s faeces contain no live genetically modified bacteria.
Figure 5. Schematic shows the initial pH sensitive and bile salt sensitive promoters used for characterization purposes. Our final aim was to achieve a construct which would produce our colicin immunity protein and synthetic colicin in response to pH when it enters the chicken stomach. In order to release the synthetic colicin our cells would lyse in response to bile salts allowing the colicins to reach their target bacteria.
This project addressed the problem of antibiotic over use in livestock by replacing antibiotics with genetically modified bacteria. To protect from further drug resistant bacterial strains, we have designed colicins which lack their cytotoxic domains and have been replaced with a multiple cloning site allowing for easy interchanging of toxins at the cytotoxic domain. In order to combat the lack of selectivity of current treatment for bacterial infections (antibiotics) the toxic warheads that may be added to the colicins have the advantage of being more specific towards strains of bacterial species. Another great benefit of this is that the natural biome within the chicken’s gastro-intestinal tract is not damaged as much as it would be by non-selective broad range antibiotics, which often wipe out healthy as well as pathogenic bacteria.
Now that we have discussed the product’s affect within the chicken and its natural gut flora, how will BactiFeed affect the environment around it? By introducing our lysis cassette we aim to prevent any live bacteria being released into the environment. This is also helpful for public perception because one of the fears associated with genetically modified organisms is their release into the environment, which, by lysing the cells, would be circumvented. To ensure that there is no genetic material, resulting from lysis, which could be taken up by other bacteria, we aimed to add a nuclease to the device. This would destroy any genetic material such as left over fragments of immunity proteins, which, if they were taken up by a pathogen would result in faster rates of resistance. The colicins are predicted to be degraded, within the chicken’s digestive system, before they are excreted.
1. 2011 Michigan State University. Antimicrobial Resistance Learning Site.
2. Public Health England, Health matters: antimicrobial resistance. www. gov.uk