Silver Medal Human Practices
Introduction
Here, we delineated the process we took throughout the course of the summer, where we met with many academics, industrial experts and employers to explore the potential of our project idea, as well as for advice as to which path to take our project. Discussions with industrial experts and employers gave us an idea of what people would be open to, in terms of the applications of our engineered bacteria, and then discussions with academics allowed us to actually put our ideas into reality.
Project initiatives
During our brainstorming sessions, we first discussed the idea of biofilms leading to Catheter-Associated Urinary Tract Infections (UTIs), such as the formation of biofilms in either the catheter or the urinary tract. With more research, another issue associated with biofilms that surfaced was the problems they caused in pipe systems. In fact, biofilms created blockages that lead to pressure build-up and were difficult to clean thoroughly, and corrosive biofilms would weaken the metal pipe itself due to Microbially Induced Corrosion (MIC).
Having planted the seed, we identified two main values for our project - Social and Moral. Antimicrobial resistance is a looming threat to humanity that could potentially make all currently available antimicrobial drugs ineffective, causing an unimaginable death toll and economic cost. The only way to stop antimicrobial resistance from developing is to stop the use of antimicrobials. We aim to achieve this goal by tackling the biggest use of antimicrobials - animal agriculture for food production.
However, it also goes without saying that the basis of our project would be rooted in good science, both in theory and practical. Thus, based on these values, we moved on to mapping a variety of stakeholders likely to be involved.
Expanding our project
While still stuck between two seemingly different applications of our project, we decided to reach out to Helix Solutions, who are experts in the field of hospital disinfection, in order to gain a better understanding of what we should consider when designing a solution for cleaning biofilms. Meetings with the National Biofilms Innovation Centre and academics from the University of Cambridge then helped us pinpoint the species to be used for our protective biofilm, as well as the use cases of B.Max.
To gain a more realistic understanding of the problem of biofilms in farms, as well as the issue of antimicrobial resistance, we reached out to other academics from the University of Cambridge with expertise in livestock animal infectious disease. Furthermore, we spoke to various farms in the UK, including the University of Cambridge’s Park Farm, Manor Farm, and Snakehall Farm, in order to learn more about the farm environment, as well as their receptivity to the idea of utilising B.Max.
Throughout the research process, we have also engaged with a wide range of academics from the University of Cambridge, the University of Southampton and Universite Paris-Saclay, some of whom also experiment with B. subtilis, and were hence able to provide much useful insight on its capability as a biofilm former, as well as troubleshoot our lab experiments. Others had much experience with corrosion measurement experiments, and the advice they offered was crucial in planning our experiments on EIS.
Why B.Max is Good for the World
It is arguably so that the most important feature of every product in the market ought to be that it is responsible and good for the world. Responsible in terms of its design and production, and good for the world in terms of its usage. As such, much consideration was put into the project to ensure that B.Max is safe, whether during its design, engineering or usage.
Firstly, B.Max is safe. We chose a GRAS (Generally Recognised as Safe) bacteria, Bacillus subtilis, that is not pathogenic to humans. Secondly, we knocked out the ability for bacteria to sporulate so the cells could never enter the highly dispersive spore state. Finally, we have designed a toxin/anti-toxin system that would kill the bacteria once it becomes planktonic (though we did not have enough time to do the actual experiments). The positive impact of our engineering product is shown by invasion assays, the results of which are available here. These factors help shape B.Max to be an inherently safe biofilm.
Moreover, B.Max would be a useful tool for the One Health Initiative proposed by the World Health Organization. One Health is “an integrated, unifying approach that aims to sustainably balance and optimize the health of people, animals and ecosystems” (“One health” 2022). Protecting animals reduces the chance of diseases being transferred from animals to humans. B.Max is an organic way to prevent bacterial infections in food-producing animals without causing antimicrobial resistance, so it is protecting the health of humans and animals without compromising the environment.
Lastly, B.Max provides an alternative for end-users who want to reduce the environmental impact of their work, especially in the case of overusing antimicrobials to combat animal diseases. Currently, antimicrobials are used for disease prevention and treatment in humans and food-producing animals at the expense of drug efficacy in the future. The application of B.Max as a protective biofilm, in addition to the biosensor for pathogenic bacteria detection, should encourage users to become more conscious of their use of antimicrobials.
Gold Medal Integrated Human Practices
Part 1: Shaping our project
Email chain with Helix Solutions - What should we consider when designing a solution for biofilm cleaning?
Date : Early July
Who : Euan Findlay (Founder and Managing Director of Helix Solutions)
Helix Solutions is one of Australia’s largest suppliers of products specialising in water treatment and disinfection. As an expert in the field of cleaning and biofilm prevention in hospitals, we wanted to understand more about the challenges they faced when designing their own products, as well as other considerations they had taken into account, which we would have otherwise not thought of.
Summary :
In emailing Helix Solutions, we asked about their current solutions and cleaning methods, and they brought up the main issues with cleaning were (a) unless completely cleaned, the biofilm would just regrow, and (b) it’s difficult to apply cleaning products to pipes for a prolonged time due to them either being vertical and/or having constant flow through them removing the cleaning products. They also mentioned that they were looking into solutions that focus on preventing the issues of biofilms forming from being a problem, rather than trying to remove the biofilms. This reminded us of an idea we’d had before, and what ultimately is the general idea of our project now: the use of positive non-harmful biofilms to take up space and prevent the growth of harmful biofilms.
Outlook :
In particular, Mr Findlay mentioned that, “one solution is to simply not try to remove but prevent the issues of it forming from being a problem.” This thus inspired us to design a solution utilising the same concept of bacterial prevention, rather than treatment. Another thing we asked about was their methods of detecting biofilms, and their only method was visual detection due to size; however, at this stage, the biofilms have grown to sizes difficult to clean and begin to cause problems. This inspired the second aspect of our project, the implementation of a biosensor that could detect the growth of bad biofilms. Click here to read more about our biosensor design.
Meeting with Dr Diana Fusco - Feasibility of using B. subtilis as our biofilm strain?
Date : 8th July
Who : Dr Diana Fusco, University Assistant Professor in Biological Physics at the Department of Physics, University of Cambridge
Where we were at :
Expanding Project Idea - Basic idea of positive biofilm to combat MIC
Having done preliminary readings, we wanted to make sure that our decision to utilise B. subtilis as our protective biofilm former is a sound decision. As Dr Fusco has done extensive work on biophysics of collective behaviour and works on models of biofilms, we were keen to learn from her.
Summary :
After an initial idea to use B. subtilis, a biofilm-forming non-pathogenic bacteria, we reached out to Dr Fusco to discuss the use of B. subtilis as a chassis. She reinforced our initial idea of B. subtilis being good at forming biofilms, but also warned us of some of its quirks.
Outlook :
The first is that B. subtilis sporulates very easily when in unfavourable conditions, and this directed us to the idea of knocking out the genes required for sporulation so our positive biofilms would be able to maintain the coat on a surface. Another being that B. subtilis, when large enough, has a tendency to pull away from the surface in some positions to create rucks. The purpose of this is not well understood, but made us consider if we could improve the adhesion of our biofilm - it turned out we didn't have enough time to explore this route fully, but you can read more about what we learned on our Discussion page. Lastly, we discussed the potential options for Dry Lab, and at the time we were considering the use of Eden (lattice) models to represent biofilm growth, something Dr Fusco had experience with. As, at the time, we were focussing on MIC, Diana suggested that rather than focusing on the 3D simulation of growth, instead focus on modelling the permeability of the biofilm to oxygen, something relevant to corrosion and somewhat novel.
Follow up :
Beyond just giving us advice on our project, Dr Fusco kindly agreed to provide us with strains that we would work on. Read more below on our discussion with Abhirub, one of the PhD students in Dr Fusco’s lab group.
Additionally, another thing we wanted to ask Diana was about a method of measuring corrosion we'd seen, Electrochemical Impedance Spectroscopy (EIS). SShe was able to refer us to some other people working at the Physics department, whom we later had a meeting with.
Meeting with Ashraf Zarkan and David Summers - MIC or Agriculture or both?
Date : Mid July
Who : Dr Ashruf zarkan, Research Fellow, Department of Genetics,researches E. coli biofilm formation; Dr David Summers, Emeritus University Senior Lecturer, Deputy Head of the Genetics Department Teaching, researches on basic plasmid biology.
Where we were at :
Expanding project idea - Basic idea of positive biofilm to combat MIC
Summary :
We met Dr Zarkan as he was the PI of one of our team members' 3rd-year project, and had very kindly offered the use of his lab to us over the summer for iGEM. We presented our project ideas about biofilms and their different potential applications, and he was interested in the use of positive biofilms and thought that their application to protect against MIC would attract more industrial interest, as opposed to the agricultural side of things. He then introduced us to NBIC, the National Biofilms Innovations Centre1, a community of biofilm researchers which allows for the sharing of information and biofilm-forming strains.
Dr David Summers from the Department of Genetics was also present during the discussion, and upon hearing our presentation, he pointed out that biofilms are often not made of solely one type of bacteria, and instead harbour a variety of microbes. As such, he was sceptical of a biofilm's ability to protect against secondary colonisation from other bacteria.
Cameron Croft, a PhD student in Dr Summers’ group, also advised that we try using safranin stain for biofilm biomass assays, instead of the normally used crystal violet stain. Cameron kindly provided his protocol for staining E. coli biofilms, which we then optimised for staining B. subtilis biofilms.
Outlook :
At this stage, we then wanted to keep the use-case of B.Max more open, instead of focusing on either one of the potential applications. After all, the basic features required of B.Max remain the same regardless of its application.
While we acknowledge Dr Summers' feedback, we decided that designing a single-species biofilm would be a good first step towards an effective solution. Given the time and resource constraints placed on our team, we agreed that designing a multi-species biofilm could be a future improvement to make B.Max more robust.
Meeting with William Green from the National Biofilms Innovation Centre (NBIC) - Is B.Max practical?
Date : 27th July
Who : William Green, Innovation and Partnership Manager of NBIC
Summary :
Having decided to keep the use-case of B.Max open, we wanted to explore the practicality of using B.Max in various situations. During our interview with Mr Green, he mentioned many potential applications of a positive biofilm. For instance, bovine mastitis on cow udders, water troughs and drinkers in farms, and corrosion in submarine containers and heat exchangers were all potential use cases of B.Max.
Outlook :
With positive affirmation by Mr Green, we are now more confident that a positive biofilm would be a useful solution for the problems we have previously identified.
Snakehall Farm visit - What is the receptivity to a synthetic biology solution?
Date : 16th August
Who : Snakehall Farm is a farming project run by the Prospects Trust in rural Cambridgeshire. It provides work, skills and training for people with additional needs, learning disabilities or autism.
Where we were at :
Understanding biofilm-related problems faced by poultry farms.
Summary :
Now exploring other ideas than MIC, we decided to look at pathogenic biofilms on farm. Our visit to Snakehall Farm opened our eyes to the workings of an organic farm, and the cleaning procedures carried out on a daily basis to maintain chicken health. Snakehall farm is not strictly a poultry farm – rather, they are a small-scale farm keeping aged or ill chickens that have been rescued from intensive poultry farms. Cleaning of the chicken coop in Snakehall occurs daily, done so by hosing down the coop, and refreshing wood shavings. Since there is no use of antimicrobials or chemical disinfectants in Snakehall, bacteria growth nonetheless represents a problem to be dealt with, thus highlighting the importance of a biological solution in fighting against zoonotic diseases in farms.
Outlook :
From our visit, we recognise that our target audience may not necessarily consist of only organic farms, with others preferring chemical methods of disinfectants due to habituation, ease of cleaning or affordability. Hence, we decided to also reach out to Manor Farm, a dairy farm in Cambridge that Snakehall Farm pointed us to, so as to obtain a wider range of opinions.
Interview with Manor Farm - Are antimicrobials typically used in farms?
Date : 13th September
Who : Manor farm Landbeach is a beef farm run by a local farmer in Cambridge since 1927.
Where we were at :
Identifying specific bacteria related problems faced by farmers.
Summary :
We got in touch with Mr Peter Hatley, the owner of Manor Farm. During our call with Mr Hatley, we learned that legislation put in place by the UK government to curb antimicrobial usage has deterred farms in the UK from using such disinfection methods. Mr Hatley is very proud that he had cared for the cattle so well that he barely had any trouble with bacterial infections. In fact, he said he had only used antimicrobials once in the past year to treat a cow with a physical injury from a fight. He also mentioned that the key to maintaining good health in cattle is plenty of outdoor time and frequent cleaning. He knew antimicrobial resistance was a serious problem and was very happy to know that we were working on it. At the end of the call, Mr Hatley also directed us to a third farm, the University of Cambridge’s Park Farm.
Outlook :
From our talk with Mr Hatley, we gathered that, on a whole, legislation is an effective way to curb the use of antimicrobials, at least proven true in the UK. Nonetheless, B.Max still has its uses, as an added defence against pathogenic bacterial invasion. However, the utility of B.Max may be more prominent in other countries where the legislation on antimicrobial use is more relaxed.
Visit to Cambridge University Farm – How much of a problem are bacterial infections on dairy farms? More on practicalities of our solution.
Date : 20th September
Who : Cambridge University Farm is a cow and sheep farm with ariable lands that demonstrates the animal welfare best practice, esplores sustainable farming and supports teaching at the Department of Veterinary Medicine at the University of Cambridge.
Where we were at :
continue exploring the problems related to actual application.
Summary :
After introducing our project to Paul Kelly, manager of the farm, he questioned the applicability of a biofilm on farm surfaces. Farm surfaces are dirty with all kinds of bacteria, and cow sheds are never thoroughly cleaned. If our biofilm requires a clean surface, it will not work on a farm.
However, Paul pointed us towards one place on his farm where the biofilm might be applicable, that is, the calf house. The calf house is a brick house where calves under the age of two weeks are placed. Calves at this stage are very vulnerable, so extra cleaning measures are taken to keep them healthy. The calf house is cleaned between each batch of calves by pressure cleaning and disinfecting, leaving the surface as sterile as possible.
When asked about antibiotic use on his farm, Paul said that he uses antibiotics for both preventatives and treatment. He believes that taking preventative measures is very important because that would prevent infections from happening. All antibiotics he uses on his farm must be ordered through a qualified veterinarian. Currently, all antibiotics that are allowed for agricultural use in the UK are non-clinically important antibiotics. Farmers need to self-report the dosage of use in units of mg of drug per kg of animal weight.
Outlook :
From Paul's words, cattle and dairy farms in the UK are well-regulated in antimicrobial use. While this is good to hear, it also means that our protective biofilm might find itself more needed in less regulated regions of the world.
Meeting with Prof. Mark Holmes - How much of a problem is antimicrobial resistance in animal agriculture?
Date : 22nd September
Who : Prof. Mark A Holmes, Professor in Microbial Genomics & Veterinary Science, researches in livestock-associated MRSA
Where we were at :
completing the picture of protective biofilm application
Summary :
We wanted to better understand how our product could be applied to the real world, and so we presented our project to Prof. Holmes, a professor of Microbial Genomics & Veterinary Science. He brought up an interesting possible distinction between antimicrobial resistance (AMR) in animals and in humans, as he's reported that the MRSA he works with in animals is not related to the MRSA found in humans in hospitals. However, he also brought up that AMR is an umbrella problem, with hundreds of resistances to different antimicrobials leading to different problems.
Outlook :
We discussed the different cleaning methods farms used that we'd seen, and he mentioned that it typically is an 'all in, all out' process, in that many farms move out all the animals temporarily whilst the farm is cleaned. This presented potentially a good opportunity for the application of protective biofilm as a post-cleaning treatment to decrease the frequency at which cleaning is required.
Part 2: Experimental planning and troubleshooting
Meeting with Dr Abhirub Mookherjee - What should we consider when designing our assays?
Date : Mid July
Who : Postdoctoral researcher in Dr Diana Fusco's lab
Where we were at :
Gathering resources
Summary :
Following our meeting with Dr Fusco, she had very kindly offered to share some bacterial strains, and in collecting them, we met with Dr Mookherjee, who streaked out some B. subtilis 3610 & 3A38 strains, and a fluorescent E. coli strain for us to use. As he works with B. subtilis, we took the opportunity to ask about the different protocols that he used specifically for B. subtilis.
Outlook :
From our meeting with Dr Mookherjee, we learned that it is best not to store B. subtilis in the fridge as the cold causes it to sporulate. Dr Mookherjee also kindly provided us with the protocols we used for transforming B. subtilis (see Protocols)
Meeting with Dr Romain Briandet, Virgile Guéneau, Dr Marie-Francoise Noirot-Gros, Hadi Jbara - Advice on biofilm assays and materials
Date : 10th August
Who : Dr Romain Briandet, Senior Researcher at the National Research Institute for Agriculture, Food and the Environment (INRAE)
Summary :
As we were inspired by Virgile and Dr Briandet’s paper on positive biofilms (Guéneau et al., 2022), we contacted them in hopes of gathering more insight on the assays we should plan for our iGEM project. In particular, they gave us helpful advice on how our assays could be carried out and which materials are most important in agriculture.
Outlook :
From this meeting, we learned that PVC and stainless steel are two of the most common materials in agriculture where pathogenic bacteria may form; therefore, we aimed to test that B.Max could form biofilms on both of these materials and also to test if B.Max would be corrosive to stainless steel.
Additionally, Dr Briandet advised that we use TSB media for our biofilm biomass assays, instead of MSgg media, as it is more appropriate for submerged biofilm assays. TSB, being rich in nutrients, would also somewhat mimic the nutrient-rich poultry farm environments.
Dr Briandet also advised utilising confocal microscopy to visualise biofilm ultrastructure and agreed that our plan to utilise a fluorescent B. subtilis strain, instead of utilising a fluorescent stain, would likely work. Whilst we didn’t have time to study the biofilm ultrastructure of our engineered strains, we did confirm that B. subtilis expressing a fluorescent protein (mScarlet) can be utilised to visualise the biofilm by confocal microscopy (see Results).
Dr Noirot-Gros warned that as the sporulation pathway and biofilm formation pathway were closely interlinked, knocking out genes related to sporulation may also negatively affect biofilm formation. This supported our previous concern that this could be the case, which is the reason why we chose spoIIE as a knockout candidate – we expected that spoIIE was sufficiently downstream of the regulatory pathways shared by sporulation and biofilm formation, such that knocking it out would not significantly negatively affect biofilm formation. This expectation fortunately turned out to be correct, as knocking out spoIIE resulted in almost complete loss of sporulation without negatively affecting biofilm formation (see Results).
Finally, Dr Briandet also mentioned that B. subtilis spore formation is important for commercial distribution of B. subtilis probiotics. Therefore, we considered making sporulation inducible, which may provide the benefits of a long-lasting non-sporulating strain, but also allow for spore harvesting for commercial distribution (see Engineering).
Meeting with Dr Andrew Grant - Should we use Salmonella and Campylobacter or Pseudomonas as model pathogen for our invasion assays?
Date : early August
Who : Dr Andrew Grant, Senior Lecturer in Bacterial Pathogenesis at the Department of Veterinary Medicine, University of Cambridge.
Summary :
Our meeting with Dr Grant then helped us in planning the assays necessary for our project. In particular, we wanted to do an invasion assay to prove that our biofilm could survive when potentially under attack from other microbes, and were initially going to use Salmonella, the zoonotic disease we were interested in. Dr Grant mentioned that even though Salmonella and Campylobacter are the main zoonotic pathogens, and both frequently form biofilms, they are often secondary colonisers of biofilms formed by “primary colonisers” such as Pseudomonas aeruginosa, which is often the first to infiltrate a biofilm and paves the way for other microbes to infect.
Outlook :
Having received this insight, we switched our sights to using Pseudomonas as part of invasion assays, rather than looking for Salmonella and Campylobacter strains as we previously did, in order to mimic a more realistic scenario for B.Max.
Presentation at the Department of Biochemistry Meeting - How do Cambridge academics think about the technical aspects of our project?
Date : 18th August
Who : Dr Martin Welch, research group leader, Department of Biochemistry, working on the control of bacterial virulence and biofilm formation in the opportunistic human pathogen; Dr Sivan Nir-Luz, Blavatnik fellow, Yusuf Hamied Department of Chemistry; Dr Ashraf Zarkan and Martin Welch’s Group.
Where we were at :
Starting work on biofilm formation assays
Summary :
We presented our work at a meet & greet at the Department of Biochemistry and had the opportunity to discuss our ideas with many academics. Dr Martin Welch, a research group leader at the Department of Biochemistry working on the virulence of Pseudomonas aeruginosa, discussed with us the competition assays we wanted to do to characterise the formation rate of our biofilm. Dr Welch was also generously able to provide us with a strain of Pseudomonas needed for our invasion assay.
Dr Nir-Luz, who's worked with B. subtilis, discussed the use of different media with us, as her experience with MSgg media was that it can be difficult to make. This corroborated with Dr Briandet’s advice.
Outlook :
Hence, we decided to switch to using TSB and grow submerged cultures, as both Dr Nir-Luz and Dr Briandet suggested, which proved to be suited to the amount we'd be using.
Meeting with Dr Svetlana Bachbut, Eleni Papafilippou and Melissa Watt - Can we do Electrochemical Impedance Spectroscopy (EIS)?
Date : 26th July
Who : Researchers at Yusuf Hamied Department of Chemistry, University of Cambridge
Where we were at :
Researching potential protocols for MIC measurements
Summary :
As our project was heading towards working either general biofilm problems or on the specific case of pathogenic biofilms on farms, we wanted to check if our biofilm was corrosive using EIS.
In the meeting, we presented our idea of using EIS to measure the rate of corrosion of metals with our protective biofilm coating. They brought up some concerns about the use of EIS, specifically the high cost of a potentiostat (the key piece of equipment), as well as the variability in the measurements themself. As EIS is very sensitive, they mentioned things such as the use of an incubator to maintain temperature, as well as it simply can be quite temperamental.
Outlook :
As such, this made us reconsider if research into MIC specifically was the most plausible; however, they did refer us to meet with Dr Jenny Zhang.
Meeting with Dr Jenny Zhang's Group- Should we EIS?
Date : 31st July
Who : Research fellows at Yusuf Hamied Department of Chemistry, University of Cambridge
Where we were at :
Researching potential protocols for MIC measurements
Summary :
We met with Dr Zhang and Dr Nir to further discuss the use of EIS. Again, they had their concerns, and also brought up a similar idea of using cyclic voltammetry. Dr Nir-Luz discussed through some of the techniques that she used in her electrichemistry research, such as the use of Quartz Crystal Microbalance (QCM) to monitor surface interactions. However, we decided the equipment requirements for these would be too difficult, and so we stuck to EIS.
Outlook :
Despite concerns about the rate of success, we thought it would be worth a try nonetheless, and decided to push forward with EIS, and Dr Zhang kindly offered the use of her potentiostats in the Chemistry department, which gave us hope. We discussed the different metals we could use, and agreed that stainless steel or steel would be good, as it's relatively easy to obtain and Dr Nir-Luz had had success with the biofilm attaching to it. The details of EIS was discussed in a later meeting with Shella Jeniferiani.
Meetings with Shella Jeniferiani Willyam - How to do EIS?
Date : Multiple times in August and September
Who : PhD student working in Dr Zhang's group using EIS
Where we were at :
Running EIS Measurements
Summary :
During our meeting with Shella, we went through what would be needed for EIS, as it has a somewhat complicated setup. The potentiostat required for EIS was provided by Dr Zhang's lab, and the metal was obtained from the Department of Engineering. However, we then needed a vessel to contain the biofilm - Shella shared that she uses a hollow cylinder of silicone, which can be placed on a metal base, and nicely has everything needed. The biofilms are able to grow on the metal base, which is then already accessible for electrical contact, and the silicone naturally seals, meaning there was no need for glue which could affect the results. It actually turns out that we had a few issues with the silicone that we ordered, so Shella suggested using nail polish to stick our silicone cylinders to the metal.
Later, after we'd done the measurements, Shella helped us with the data analysis, as the initial software that we were using, AfterMath3, was unfortunately not working with our data. Shella was able to direct us to using IviumSoft4, in which we were able to able to analyse the data we'd collected.
Closing the loop
By speaking to a wide variety of stakeholders, and gathering a range of opinions from different organisations in the same field, we managed to close the loop between what is needed from B.Max and what we have engineered. However, having spoken to various experts, we also identified possible ways to further improve B.Max (see Engineering).