In order to refine our project based on insights gained through our Human Practices efforts, it was essential to create a concise and
comprehensive summary. This summary is used to highlight the contribution of each meeting, articulate its impact on project adjustments, and
outline our next steps. We chose to employ the AREA framework, originally introduced by iGEM Exeter 2019, to process the results of our
stakeholder interviews. This structured approach ensured that we made the most of the valuable knowledge we've gathered, resulting in further
project optimization.
Meeting #1
Meeting Associate Professor Molecular Evolution 1
Date: 07-06-2023
Stakeholder: Associate Professor Molecular Evolution
Description: This Associate Professor from the University of Groningen has experience with working with phages and designing phages. We
wanted to talk to this professor about the design and experimental procedures regarding a phage treatment system for our project.
Contribution: We gained knowledge in how the M13 phage works. M13 serves as a non-lethal, non-toxic bacteriophage used for the delivery
of toxin genes or genes that disrupt biofilms, and possesses an inherent amplification function. The biosensor cell should be designed to
detect specific analytes and should incorporate two plasmids. The first plasmid houses the p3 gene responsible for phage production, which
undergoes exponential amplification. This gene is regulated by a promoter sensitive to the analyte being monitored. The second plasmid
contains either a cell-toxic gene or enzymes that degrade biofilms, rendering the biosensor bacterium safe. The analyte of interest may
include quorum-sensing molecules typically found during biofilm formation.
To confine the biosensor, it can be immobilized on a porous material that permits the passage of phages and analytes but restricts the
movement of the biosensor. For reporting infections, gold plates coated with streptavidin can be employed, and the p3 gene can be tagged with
a Strep-tag. This setup is connected to a device that precisely measures thickness.
When virus particles bind to streptavidin through the Strep-tag, it induces a change in measurement, generating an electric signal. A device
analogous to a glucose monitoring device, utilizing a flexible, partially gold needle for streptavidin and a porous material for the
biosensor, can be used for this purpose.
Adjustments: We are not making any definitive adjustments as of now, but will look further into the working principle of the M13 phage
and if it will be feasible for us to use.
Our next steps:
Look into working principle of M13 phage.
Look into possibility of delivering a gene that can degrade the biofilm matrix, look into possible enzymes.
Type of quorum sensing molecule.
Look into design specifics of the biosensor device, or focus more on the proof-of-concept idea of using a synthetic biology solution,
including the use of the phage treatment system.
Meeting #2
Meeting Associate Professor Orthopedics Department Principal Investigator at Regenerative Medicine Center
Date: 26-06-2023
Stakeholder: Associate Professor Orthopedics Department at University Medical Center Utrecht (UMCU) and Principal Investigator at the
Regenerative Medicine Center Utrecht.
Description: This stakeholder is Associate Professor at the Department of Orthopedics, University Medical Center Utrecht (UMCU) and
Principal Investigator at the Regenerative Medicine Center Utrecht. This person has a background in material science engineering. We wanted to
talk to this stakeholder about the state of the art of biosensors for medical implants.
Contribution: During our discussion, we delved into our project and explored the current landscape of biosensors for implant
applications. Managing biofilms presents numerous challenges, mainly due to the protective function of extracellular polymeric substances
(EPS) shielding bacteria from antibiotics. Furthermore, there exists a critical 4-6 hour post-surgery window during which planktonic bacteria
endeavor to adhere to implant surfaces. It is within this time frame that biofilm formation can be prevented through material-based techniques
such as the release of silver nanoparticles, utilization of nanopillars, and engineered nano-structures. Integration of these strategies
directly into the implant design is an important consideration. The primary challenge lies in identifying a method that exhibits bactericidal
properties without impeding implant assimilation.
Bacterial colonization can occur beyond this initial window. Additionally, there are ongoing advancements in electrical biosensor systems. To
his knowledge, there is currently no system capable of both detecting biofilm formation and simultaneously activating a treatment response. To
ensure safety and compatibility, the materials employed must meet biocompatibility criteria, excluding substances like lead and cobalt.
He is willing to collaborate with us or provide supervision. He shared three research papers that we can summarize and submit to him. This
literature will assist us in refining our research question and experimental design. He has also offered to provide feedback on our
methodology and guide us in formulating a set of experiments. In September, he plans to be in the Netherlands for several PhD defenses, and we
have the opportunity to meet with him in person.
Adjustments: Since we are not looking into new materials to prevent biofilm infections this field is not directly relevant for our
project but it was interesting to gain more knowledge in the field of biofilm prevention and the different options.
Our next steps:
He sent us a couple of review papers about biosensor systems for biofilm detection.
We will summarize the review papers and send it to him.
Unfortunately, we didn’t hear back from him.
Meeting #3
Meeting Associate Professor Molecular Systems Biology, Systems Biology of Signaling Networks
Date: 12-07-2023
Stakeholder: Associate Professor in the Molecular Systems Biology unit of the RUG, Systems Biology of Signaling Networks.
Description: This stakeholder is Associate Professor in the Molecular Systems Biology unit of the University of Groningen, Systems
Biology of Signaling Networks Department. We wanted to talk to this stakeholder about bioluminescence, get advice on the use of luciferase and
gain some information about some electronics parts we can use to convert the signal to an electrical output.
Contribution: The discussion covered bioluminescence, luciferase, and electronic applications. One key consideration was the necessity
of a substrate for luciferase and its practical applications. We talked about the feasibility of using a chemical signal rather than light,
discussing issues like point measurement limitations, the diffusion of quorum sensing molecules, and detection sensitivity. The choice between
single-use and multi-use systems was discussed, along with the viability of the biosensor's bacterial components. The idea of creating a
predictive model for interactions between the biosensor and phages with biofilms was proposed.
Fluorescence, while bright, was considered less practical due to excitation requirements and signal acquisition challenges. It was noted that
fluorescence was easier to implement in oxygen-rich environments. Optic fibers and the need to identify suitable wavelengths for signal
transmission were also emphasized, along with photodiodes and amplifiers for signal detection. The aim is to find luciferase variants that
function without external substrates. The proof of concept centered on engineering cells to respond to specific inducers, characterizing their
responses, and quantifying emitted light while minimizing noise in signal detection.
The stakeholder told us that there is a workshop in a building on the university campus that can help us with building the electronic device.
Adjustments: There are several considerations that we need to consider in our project and in the design of our electronic device. We
need to look into bioluminescence, rather than fluorescence. We decided to focus on the proof-of-concept of engineering the cells for the
detection and treatment system rather than designing a complete functional device as it is very complicated and probably too much for the
time-scope we have.
Our next steps:
Look into a type of luciferase that functions without an external substrate.
Single use vs. multiple use system.
Look into optic fiber cables Read about diffusion of quorum sensing molecules Focus on proof-of-concept of engineering the cells for our
system, rather than focussing too much on the functional biosensor part.
Meeting #4
Meeting Professor UMCG, Department of Biomedical Engineering
Date: 13-07-2023
Stakeholder: Professor UMCG (University Medical Center Groningen), Department of Biomedical Engineering.
Description: This stakeholder is professor at the UMCG (University Medical Center Groningen) in the Department of Biomedical
Engineering. Their area of expertise primarily focuses on biomaterial-related infections within the human body. Specifically, their research
centers on examining the impacts of modified biomaterials on bacterial adhesion and the formation of biofilms. We wanted to talk to this
stakeholder to gain more insight into the problem of biofilm formation and the challenges biomedical engineers face with regards to
engineering things to prevent their formation or to treat them.
Contribution: Antibiotic treatments face limitations as they primarily act on the surface. Extensive research has been devoted to
developing coatings, including polymer brushes and antibiotic coatings, although the latter depletes over time. Effective coatings must be
both hydrophilic and smooth. Some materials can not only prevent biofilm formation but also facilitate antibiotics' penetration from below.
Researchers are exploring micelles for treatment, although detecting biofilm formation remains challenging. Techniques such as labels and PET
scans prove complex, and fluorescent probes, tested in mice, are challenging to detect through human skin. Targeting the location for
antibiotic treatment is crucial.
Biofilm risks include issues with implants and dental procedures. Stakeholders, such as implant manufacturers, face significant costs during
the clinical phase, and testing on high-risk patients is generally prohibited. Moving away from biofilm prevention with materials to
alternative treatments is due to the durability of prosthetics and the wear and tear on materials over time. While dispersing biofilms is
appealing, it carries the risk of bacteria relocating and introducing different toxic genes. A non-specific approach to biofilm degradation
allows for the treatment of various biofilm types.
The bioluminescence-based biosensor design has a localized detection limitation, addressing only one side of the implant. Deciding the
proximity to the implant that requires sensing poses a challenge. Although this technology holds importance for all patients, it's
particularly crucial for immunocompromised or chronically ill individuals. Healthy patients typically have robust immune responses, reducing
the frequency of problems. The question of whether this technology should apply to all patients arises, with the possibility of focusing on
revision surgeries, especially for individuals with prior infections, where the risk of biofilm infections is notably higher.
Adjustments: We have redefined our focus group to primarily immunocompromised individuals that are at a higher risk of developing
biofilm infections as a result of their medical implant. Sensing biofilm formation in the body might be complicated using a
bioluminescence-based biosensor as there is a detection limitation, as only one side of the implant will be addressed. We still think we
should focus on the proof-of-concept synthetic biology part of the project since it will be very complicated to design a device that already
potentially could be used as a complete biosensor for biofilm formation within the time that we have for the project.
Our next steps:
Focus more on specific target group, will help with conveying the story around our project and the background and inspiration
corresponding to our project.
Decide whether to stick with bioluminescence as primarily a proof-of-concept of the synthetic biology system.
We could look into the properties of a complete biosensor system as well if we have more time, but we are probably not able to actually
design such a complicated system.
Phage treatment involving degradation of the biofilm matrix can make the bacteria more sensitive to subsequent antibiotic treatment.
We need to keep the context and the whole picture of treatment in mind.
Meeting #5
Meeting MSc Student with experience with optogenetics in mice
Date: 14-07-2023
Stakeholder: MSc student with experience with optogenetics
Description: This stakeholder is a MSc student who has experience with performing optogenetic experiments in mice. We wanted to talk to
this stakeholder to gain more insight into how optic fibers work.
Contribution: We learned that the optical fibers are very small and it's crucial to determine their detection threshold. Additionally,
we must address motion-related noise interference. An important consideration is the optimal duration for the biosensor's activity. It's worth
exploring the prevalence of implanted optical fibers. We need to devise methods for maximizing light measurement efficiency while ensuring the
durability of the system and safeguarding against damage during movement. Evaluating how external light sources may impact the signal is also
important. Investigating the diffusion patterns of c-di-GMP is essential. Adjustments: We realized that this system would probably not
be durable enough for a practical application as a wearable biosensor device for the detection of biofilms. However, if we manage to build
such a system we could still do it as a proof-of-concept for an electric device that can convert the bioluminescence output into an electrical
signal.
Our next steps:
Look into how to design a device with optical cables Diffusion of c-di-GMP.
Durability of the system.
Meeting #6
Meeting with Orthopedic Surgeon
Date: 17-07-2023
Stakeholder: Head Department Orthopedics UMCG (University Medical Center Groningen), Orthopedic Surgeon.
Description: This stakeholder is head of the Orthopedics department at the UMCG and orthopedic surgeon. We wanted to talk to this
stakeholder to gain more insights into the problem of biofilm formation on orthopedic medical implants.
Contribution: In implant surgeries, around 2% of patients develop infections related to biomaterials. Annually, there are approximately
70,000 primary implant surgeries, in addition to 50,000 revision surgeries that carry an infection risk. This leads to more than 1,000 to
2,000 patients suffering from infections each year, with prosthetic implant-related mortality rates rivaling those of breast cancer. In severe
cases, amputation becomes necessary. The cost of a primary implant is around 8,000 to 9,000 euros, with hospital treatment and recovery
expenses totaling 35,000 to 40,000 euros. Extensive antibiotic use in these cases contributes to the growing problem of antimicrobial
resistance, potentially making implant-related infections a leading cause of death worldwide by 2050. Unfortunately, developing new
antibiotics is often unprofitable for pharmaceutical companies.
Early infections manifest within three months of primary surgery and exhibit symptoms like prolonged wound leakage, redness, and wound
infection. Detecting chronic inflammation is more challenging; practitioners may try to aspirate fluid for culture testing, but capturing
bacteria in biofilm mode is difficult. The use of pH monitoring, as the environment becomes more acidic in the presence of biofilm, has been
considered. Multifaceted diagnostics, including clinical results, radiographs, blood inflammation levels, and the combination of various
factors, can enhance the probability assessment of the condition.
Regarding biosensors, there is a risk of infection if they have direct contact with the joint and the external environment. He suggested some
individuals we could talk to. Practical considerations for biosensors include their small size, durability, smoothness, and trauma safety.
They should also be constructed from materials that do not degrade within the body. A system with wires is not very durable.
This stakeholder supports the project, recognizing its societal relevance in the context of an aging population and the increasing reliance on
artificial and biomaterials to reconstruct aging or damaged body parts.
Adjustments: We should use the numbers and background regarding healthy aging for the story around our project. We realized again it
will be hard to design an actual wearable design. We need to decide whether we want to actually design a wearable device which will be
difficult or if we try to build something we can build with the available resources and knowledge we have. We could look further into
possibilities in literature but it will be too complicated for this project to design an actual wearable device. We decided we want to make a
survey to collect experiences from (ex-)patients with biofilm infections on their medical implants.
Our next steps:
Look into practical considerations of wearable biosensor devices.
Contact person this stakeholder mentioned for how we could reach (ex-) patients with a survey.
Meeting #7
Meeting Associate Professor Molecular Evolution 2
Date: 10-08-2023
Stakeholder: Associate Professor Molecular Evolution
Description: This Associate Professor from the University of Groningen has experience with working with phages and designing phages. We
already talked to this professor about the design of a phage system on 07-06-2023. We wanted to talk to the professor again to discuss more
details about the phage system.
Contribution: We learned that we should use another strain for expressing the phage, we cannot use E. coli. We talked more about the
design of the phage, using an M13 phage for the phagemid and a helper phage. We want to use the phage for delivery of a gene that can degrade
the biofilm matrix. The M13 phage is a filamentous phage, and it is more often used for phage display. Delivering a cargo is poorly explained
in literature. The professor looked at the approach we designed for the phages and thinks we are on the right track. He will get us in contact
with e PhD’er in his group who has worked with the phages, she can also tell us more about the experimental procedures.
Adjustments: We cannot use E. coli for expression of the phagemid. It’s better to order the whole phagemid and not construct it
ourselves.
Our next steps:
Meet with contact person to get TG1 cells, phagemid plasmids and experimental procedures for working with these constructs and cells.
Meeting #8
Meeting with Detact Diagnostics
Date: 11-08-2023
Stakeholder: Detact Diagnostics
Description: Detact Diagnostics is a start-up company working on various bacterial, viral and fungal detection solutions. They are
developing multiple variations of a product that is able to detect bacterial infections via the presence of bacterial peptidases. We wanted to
talk to them specifically about their system for detection of bacterial growth - such as biofilm - in the synovial fluid, SynoTact.
Contribution: Detact Diagnostics has developed a point-of-care diagnostic test for detection of bacterial infection. They are testing
and developing various applications of their system, amongst others for detection of periprosthetic joint infections (PJIs). They are
simultaneously developing small-sized optometric-based screening devices that allow anyone with a device to test samples. Enabling a quick (no
incubation time) and simple test just in the doctor’s office providing data in minutes. The test is based on detection of bacterial peptidase
presence in the synovial fluid. If bacterial presence is established in the synovial fluid, this needs to be acted upon as soon as possible to
prevent more severe consequences such as revision surgery. The procedure requires collection of synovial fluid with a needle.
Furthermore, Detact Diagnostics was able to tell us the relevance of our mission statement. Initially Detact Diagnostics was founded by a
University Medical Centre Groningen affiliated Trauma Surgeon, Dr. Joost Gazendam. Dr. Gazendam saw the rising problem of prosthetic joint
infection leading to the establishment of Detact Diagnostics. Besides sharing the problems faced by professionals in the field, Detect
Diagnostics also shed some light on the process, and difficulties, of putting a medical test and a medical device on the market.
Adjustments: It was very interesting to hear about the story of Detact Diagnostics, a company that is tackling a largely related
problem with a radical different approach. Detect Diagnostics pointed out the difficulties of getting medical devices and/or tests approved
for (European) markets. While in our proof-of-concept phase, this did not result in any direct changes, but it is something to take into
account with potential progression of our project. Detact Diagnostics also suggested other stakeholders and institutions we could talk to. The
meeting didn’t result in any direct adjustments as their system is quite different from what we are designing. Or their contributions are to
be taken into account in further stages of our project, which will be outside the scope of the current iGEM project.
Our next steps:
If we do want to do something with entrepreneurship we could contact them again.
Look into the possible issues we can run into while introducing our sensor into the body.
Be warned about the difficulties and the lengthiness of the process of getting medical tests and/or devices approved on (European)
markets.
Meeting #9
Meeting PhD’er with experience with phages
Date: 16-08-2023
Stakeholder: PhD’er with experience with phages
Description: This stakeholder is a PhD student with experience in working with phages. We wanted to talk to her to get TG1 cells and
phagemid plasmids, and to get information about the corresponding experimental procedures.
Contribution: The PhD’er gave us TG1 cells and two phagemid plasmids, along with the Benchling plasmid maps and cloning protocol. She
gave us a good source in which the procedures are described. When we are going to work with phages we need to buy Virkon for safety reasons
for proper disinfection.
Adjustments: We decided to use these constructs as we got access to these and we now know the corresponding experimental procedures.
Our next steps:
Read literature sources and look into experimental procedures for phagemids.
Get overview of cloning strategy.
Look into how to incorporate cargo (enzyme that can degrade matrix of biofilm).