Team:TU-Eindhoven/Human Practices

Integrated Human Practices

“Things grow stronger when you integrate” – Austin Freeman

For our human practices, we spoke to a wide variety of IBD stakeholders, including doctors, patients, and field-specific experts and companies. Together with them, we looked into the societal, medical, entrepreneurial, legal, and ethical considerations of our project. In this way, we created feedback loops involving reflection, responsibility, and responsiveness between IBDetection and the real world. On this page, the most important insights of the conversations with the different stakeholders and how they shaped our project are reported within eight categories.

The Science

The first of these categories is The Science, in which we expand upon how we reached the literature that was eventually implemented in our project. This can be traced back to the beginning of our project, where we had in-depth discussions with our supervisors on novel new strategies in the field of synthetic biology and problems with high impact on society. In this section, we will walk you through the integration of their feedback into our ideation phase, culminating in the establishment of our initial project goals and project design.

Human Practices

Integration

Supervisors: prof.dr. Maarten Merkx, prof.dr.ir. Luc Brunsveld, and prof.dr.ir. Tom de Greef

Every week, we came together with the supervisors to discuss our progress, and brainstorm ideas. After weeks of deliberation and research, the supervisors provided us with an article describing a novel method to genetically express protein-based gas vesicles. These gas vesicles could be detected and ruptured using conventional ultrasound equipment, pointing to the huge potential for application in cell-based imaging. Our next step was to search for a suitable and high-impact application for this technique. Since ultrasound is a known technique with deep-tissue penetration, this method could be applied for the imaging of tissue deep inside the body, without having to enter it. This pointed us in the direction of diseases afflicting the abdominal region of patients, as this is currently performed with measuring equipment inside of the body. In particular, Inflammatory Bowel Disease (IBD) stood out, which will be further touched upon in The Need.

Article - Protein based gas vesicle [1]

The authors of this paper introduced the term acoustic reporter genes (ARG); genetic constructs that allow bacterial gene expression to be visualized in vivo using conventional ultrasound equipment. These genes are derived from cyanobacterium Anabaena flos-aquae and Bacillus megaterium. In contrast to existing methods for imaging, mostly based on optical reporter genes with limited deep tissue performance, ultrasound is a widely used inexpensive method that has a deep tissue penetration and high spatial resolution. The genetic constructs are based on gas vesicles, i.e. gas-filled protein nanostructures. Gas vesicles are defined as protein shells with sizes around 200 nm that enclose hollow interiors, and where dissolved gases can permeate while water is excluded. Bourdeau et al. found that these gas vesicles can scatter sound waves due to the difference in acoustic impedance between the shell and the gas, and thereby produce ultrasound contrast which makes it possible to measure the signal with ultrasound.

Furthermore, they tested the possibility of ARGs to serve as an output signal for engineered genetic circuits by placing the ARG proteins under the control of an IPTG controllable T7 promoter. They showed that ultrasound contrast could be observed in vivo 4h after IPTG induction and increases during the 22h period of culturing. To confirm that the ultrasound contrast is from the presence of gas vesicles, they apply acoustic pulses with amplitudes above the critical collapse pressure of the gas vesicles. Thereupon, the proteins collapse immediately, the gas contents dissolute, thereby eliminating the ultrasound contrast. Now, a second image is made and the two images can be subtracted from each other, keeping only the scattering of the gas vesicles. They also found that these different ultrasound waves do not affect cell viability.

With promising in vitro measurements, they also set out an experiment to test the detectability of the ARG protein in vivo whereby the bacterium is localized inside the colon. They have done this in mice and used E. coli Nissle 1917, which can colonize inside the mammalian gastrointestinal tract and have been used clinically in humans to treat infection and inflammatory bowel conditions. The resulting ultrasound images showed that the ARG proteins are expressed inside E. coli Nissle 1917 cells. So, these results establish the ability of ARG proteins to make genetically engineered microorganisms visible non-invasively in deep tissue. Furthermore, it demonstrates the advantage of ultrasound relative to optical imaging in terms of spatial localization within deep organs.

Article - IBD sensing bacteria [2]

The complexity and relative inaccessibility of the gut environment makes realistic in vitro gut models challenging. Consequently, the processes in gut pathways remain poorly understood. This paper presents a novel way of analyzing gut pathways by showing the potential of genetically engineered sensor bacteria. These sensor bacteria enable control of the expression of reporter genes and thereby non-invasive measurement of gut metabolites in the colon and fecal samples.

Gut sulfur metabolism is linked to inflammation. Sulfate-reducing bacteria present in the colon produce hydrogen sulfide (H₂S) from oxidized sulfur species derived from the host. At high concentrations, H₂S can be toxic to host cells, and thereby prevent oxidative phosphorylation. Host enzymes can detoxify H₂S by converting it to thiosulfate. Furthermore, it is shown that reactive oxygen species produced by the host during inflammation convert thiosulfate to tetrathionate. Therefore, colonic thiosulfate and tetrathionate levels can be related to inflammation, such as IBD.

In this paper, they present two sensor mechanisms with specific orthogonal pathways to sense tetrathionate and thiosulfate. The sensor mechanism of thiosulfate is a novel bacteria while the sensor mechanism of tetrathionate is further optimized. To report the sensing of these molecules, a cascade is activated which will result in the expression of recombinant super folder Green Fluorescent Protein. The fluorescence is measured using flow cytometry equipment.

As will be explained in The Science integration, tetrathionate is the used marker in our project. Therefore some specifications about the tetrathionate sensor will be given. After optimizing the tetrathionate sensor, they examined the sensitivity and specificity as these properties are crucial for engineered sensors. The sensors showed a half-maximal activation (EC50 at 50 ± 3 µM. To determine the specificity, they tested the sensor versus a wide range of molecules. The sensor did not respond to any of these molecules, demonstrating a high specificity. This system was shown to be responsive in E. coli BL21 (DE3) and E. coli Nissle 1917, under aerobic and anaerobic conditions. This data indicates that genetically modified bacteria can sense and respond to gut metabolites correlated to inflammation. Thus, this illustrates the potential to identify IBD in living mammals without invasive testing.

Integrating the two aforementioned papers, we envisioned a bacterium, E. coli BL21 (DE3) for our proof-of-concept, that contains a merged version of the TtrS/R sensing system and the ARG proteins. We chose to integrate the tetrathionate system over the thiosulfate system because it was written that tetrathionate is the resulting metabolite produced during gut inflammation. Furthermore, the mechanism for the detection of this metabolite is sensitive, highly specific, and combinable with the gas vesicles. The TtrS/R sensing system will detect the intestinal inflammation marker tetrathionate. Upon tetrathionate detection, the expression of ARG proteins will be activated, resulting in the production of gas vesicles which can be subsequently detected using ultrasound technology. This will lead to a specific, and non-invasive diagnostic method for IBD using ultrasound technology. A more detailed explanation of the mechanism can be found on the Proof of Concept page.

References

1. Bourdeau, R. W., Lee-Gosselin, A., Lakshmanan, A., Farhadi, A., Kumar, S. R., Nety, S. P., & Shapiro, M. G. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature, 553(7686), 86–90. https://doi.org/10.1038/nature25021

2. Daeffler, K. N., Galley, J. D., Sheth, R. U., Ortiz-Velez, L. C., Bibb, C. O., Shroyer, N. F., Britton, R. A., & Tabor, J. J. (2017). Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular systems biology, 13(4), 923. https://doi.org/10.15252/msb.20167416

The Need

After weeks of deliberation, reading papers, and consulting experts in the field of healthcare, we established our project goal at the time: a specific and non-invasive diagnostic method for IBD by use of ultrasound. To identify the need in the field of IBD, we searched for the worldwide and national incidence as well as the total costs of IBD. Furthermore, we talked to gastroenterologists and IBD patients to clarify the current IBD diagnostic tract and identify its shortcomings, resulting in further refinement of our goals and project.

Human Practices

Integration

Article - IBD incidence [1]

Annually, over 6.8 million people suffer from IBD and an increasing trend of IBD cases has been reported worldwide. Furthermore, there is a relationship between the IBD incidence and the socio-demographic index (SDI), which is an indicator of the development status of countries. High SDI locations have the highest IBD age-standardized incidence rate compared to low SDI locations. However, in the past few decades, a rapid rise of IBD occurrences has been documented in newly industrialized countries of Asia, South America, and the Middle East. Moreover, some of these countries have populations exceeding 1 billion people. These large populations in combination with urbanization and westernization significantly increase the economic and social burden of the government, society, and health care systems, making IBD a worldwide problem.

Article - IBD costs [2]

In this paper, the direct and indirect costs of Danish IBD patients are evaluated. The total IBD costs are the summation of the direct and indirect costs. For Crohn’s disease and Ulcerative Colitis, these total costs are 11.100 euros and 9.800 euros respectively per patient per year. The direct costs consist of the care and treatment of IBD patients. This includes surgery, hospitalization, infusions, biologicals, and diagnostic procedures. The indirect costs consist of paid sick leaves at work, periods of unemployment with the resulting social security benefits payments, and days of absence. The diagnostic procedure, e.g. the endoscopy, costs are estimated at 800 euros per patient per year.

Crohn & Colitis NL

Crohn & Colitis NL is a Dutch voluntary organization that is dedicated to finding a cure for Crohn's disease and Ulcerative Colitis and improving the quality of life for those affected. On their website, they mention that 1 in 200 people suffer from IBD in the Netherlands which equals over 90.000 people. Furthermore, every year more than 300 young children are diagnosed with Crohn’s disease or Ulcerative Colitis and this number keeps rising disturbingly fast.

Marieke Pierik - Gastroenterologist

Marieke Pierik, a gastroenterologist working at the Maastricht University Medical Centre, explained to us how IBD is currently diagnosed. She told us that this consists of an endoscopy. During an endoscopy, the organs inside the body are visualized by the use of an endoscope, which is a long, thin, flexible tube that has a light and a camera at one end. When the intestine needs to be visualized, the endoscope is inserted through the bottom, resulting in cramping and pressure. Additionally, Marieke told us all the downsides that come with an endoscopy procedure. An endoscopy takes around 30 minutes and one gastroenterologist together with two nurses is needed, making it a complex, time-consuming, expensive, and invasive procedure. Furthermore, she told us that the preparation of the procedure, fasting and taking laxatives, are very unpleasant for the patient.

IBD patients - Interview

Several IBD patients shared their experiences and feelings related to IBD during interviews with us. During the IBD patient interviews, we asked them to share annoying IBD real-life situations. One patient told us that just going to a restaurant or ordering some food is very difficult due to the highly sensitive gut. Another patient explained that you continually need toilets nearby. Going to the city center where no toilets are present is therefore almost impossible. We also asked the patients to elaborate on the way IBD is diagnosed, the endoscopy. The patients told us that as a preparation for this procedure, strong laxatives should be used. Furthermore, the day before the examination, no food can be consumed. The complete ‘cleaning’ of the colon is the biggest issue that comes with the diagnosis of IBD. When we asked them if they would rather have another way for IBD diagnosis, the overall answer was: “It would be more than desired to replace the endoscopy for a more pleasant procedure for which no laxatives are required”.

The general public - Survey

The willingness to undergo an endoscopy procedure was analyzed by use of the self-made survey with our partner iGEM team BOKU Vienna. The question: “What is your feeling about getting an endoscopy?” was answered 344 times by people all over the world of which nine have been diagnosed with IBD. The answer options were: I do not mind, I prefer not to, and neutral. 44% of all the people and 22% of the IBD patients answered the question with preferably not, pointing out the need to improve the current way of IBD diagnosis (Figure 1).

Figure 1: Questionnaire results for undergoing endoscopy examination.

In the last few years, the IBD incidence is increasing rapidly. Worldwide, over 6.5 million people have been diagnosed with IBD of which 90.000 people are in the Netherlands. This growth in incidence leads to an increasing burden on both social and economic fields, especially in the newly industrialized countries. The total yearly costs per IBD patient, i.e. direct and indirect costs combined, are approximately 10.000 euros. As a consequence, worldwide, the total costs for IBD exceed over 65 billion euros.

The diagnosis of IBD consists of an endoscopy. From the patient interviews, it became clear that endoscopy is very unpleasant for the patient. In particular, the complete “cleaning” of the colon; strong laxatives are used from the day before the procedure. As a result, the patient needs to be near a toilet all day long. Furthermore, the endoscopy itself is painful as a tube is inserted into the rectum resulting in cramping and pressure.

According to the gastroenterologist, an endoscopy is seen as a complex, time-consuming, expensive, and invasive procedure. This is because three people, of which one gastroenterologist and two nurses, are needed for a 30 minutes procedure. Furthermore, the costs of the endoscopy procedure are estimated at 800 euros.

We, therefore, decided to expand on our project goals by tackling the complex, time-consuming, expensive and invasive nature of the endoscopy with a solution that is non-invasive, specific, fast-imaging, cost-effective & easy-to-use diagnostic method for IBD by use of ultrasound. A detailed explanation of these goals can be found on our Description page.

References

1. GBD 2017 Inflammatory Bowel Disease Collaborators. The global, regional, and national burden of inflammatory bowel disease in 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Gastroenterol Hepatol. 2020 Jan;5(1):17–30.

2. Lo B, Vind I, Vester-Andersen MK, Bendtsen F, Burisch J. Direct and Indirect Costs of Inflammatory Bowel Disease: Ten Years of Follow-up in a Danish Population-based Inception Cohort. J Crohns Colitis. 2020 Jan 1;14(1):53–63.

The Medical Field

We wanted to discuss our project goal with stakeholders in the field. We asked IBD patients and the general public if they would prefer ultrasound measurements over an endoscopy, to see if there would be interest in our product. Furthermore, we talked to gastroenterologists working in the medical field. In this way, we wanted to see where improvement can be made and hoped to receive useful feedback that could be incorporated into the product design.

Human Practices

Integration

The general public - Survey

Together with our partner iGEM team BOKU Vienna, we made a general public survey to determine the willingness to undergo an endoscopy and an ultrasound measurement. 344 people from all over the world filled in the survey of which 4% (Figure 2) would prefer not to undergo an ultrasound measurement while 44% (Figure 1) would prefer not to undergo an endoscopy. Of the people who completed the survey, 9 were diagnosed with IBD. In this subset, nobody (Figure 2) rejects the ultrasound measurement while 22% (Figure 1) prefer not to undergo an endoscopy. This made clear that our technique would be highly preferred over the current way of diagnosing by both the general public as well as IBD patients.

Figure 1: Questionnaire results for undergoing endoscopy examination.

Figure 2: Questionnaire results for undergoing ultrasound examination.

IBD patients - Interview

Six IBD patients were interviewed and asked to elaborate on their experiences with the endoscopy and the preparation for this procedure. Interestingly not the endoscopy, but the preparation by use of strong laxatives and fasting seemed to be the biggest issue concerning the diagnosis of IBD. This is because, during the endoscopy procedure, a sedative is used which makes it not very painful for the patient. However, in preparation for the endoscopy 2L of laxative has to be consumed, which is a very thick liquid that tastes unpleasant due to the artificial flavoring that is added. All patients that were interviewed indicated that it would be a huge improvement if the use of laxatives can be avoided. When we confirmed that for the ultrasound measurement no laxatives and fasting were required, the patients were very excited. Furthermore, we asked the patients if they would ever consider the intake of GMOs for the diagnosis of IBD. On this, they answered unanimously yes under the proviso that safety is guaranteed and a clear explanation is given.

Marieke Pierik - Gastroenterologist

We demonstrate our project goal to Marieke Pierik, a gastroenterologist working in the field of IBD. She immediately mentioned that endoscopy is the golden standard for the diagnosis of IBD. This is because an endoscopy gives direct visualization of the condition of the gastrointestinal tract, e.g. redness and blisters, and biopsies can be taken to distinguish between Crohn's disease and Ulcerative Colitis. Therefore, improving the endoscopy will be very difficult. However, she told us that the monitoring of IBD, i.e. controlling disease flare-ups, could be improved. Marieke told us that early detection of flare-ups is important to prevent scar tissue formation as a result of perforation of the intestinal wall. Furthermore, she elaborated on the current way of IBD monitoring. The monitoring of IBD is based on two components: 1) the point of care (POC) fecal calprotectin home test, e.g. IBDoc, performed on the patient's stool sample, and 2) a periodic survey which consists of disease-related and general questions. Fecal calprotectin levels are increased when there is inflammation of the intestine. However, Marieke told us that fecal calprotectin levels are also influenced by other factors such as hemorrhoids, making it a nonspecific biomarker. Therefore, often, unnecessary invasive follow-up endoscopies are taken.

Mark Lowenberg- Gastroenterologist

Mark Lowenberg is a gastroenterologist at the Academic Medical Center in Amsterdam. He also told us that endoscopy, in his opinion, is the best way to diagnose IBD. The endoscopy gives direct visualization of the seriousness and location of the inflammation. He, similarly to Marieke, suggested focusing on the monitoring of IBD. Interestingly, Mark already uses ultrasound measurements (without GMOs) in addition to the endoscopy for IBD diagnosis. He told us that an ultrasound measurement gives additional information, e.g. wall thickness of the intestine, that can indicate inflammation. Therefore, he emphasized that we should clarify the added value of our proposed technique. Furthermore, he gave us two additional options on which our technique can be applied; therapy-response monitoring and differentiating between IBD and irritable bowel syndrome (IBS).

Maurice Lutgens- Gastroenterologist

Maurice Lutgens is a gastroenterologist at the Elizabeth-TweeSteden hospital in Tilburg. During the meeting, we discussed the fecal calprotectin test. He told us that this test is a quantitative measure of neutrophil flux to the intestine, which occurs during intestinal inflammation. However, the test is nonspecific and the location of the inflammation cannot be extracted. Therefore, he thinks that our technique would be a good addition to the POC fecal calprotectin home test. When the fecal calprotectin test shows a positive result, our technique can be used to verify if it is due to inflammation inside the intestine or not. This will reduce the number of unnecessary endoscopies taken due to the nonspecificity of the fecal calprotectin test. Furthermore, he emphasized that monitoring of inflammation flare-ups is very important. Scar tissue formation due to untreated inflammation is correlated to the risk of colorectal cancer development. Therefore, the quantitative analysis of our technique can be used to indicate the seriousness of the disease and subsequently do medicinal dosage adjustments.

We also asked Maurice if we could use our technique to distinguish between IBD and IBS as Mark suggested, however, he told us that the fecal calprotectin test works accurately enough for this distinction. Furthermore, ultrasound measurements for the diagnosis of IBD are not used in Tilburg as trained operators are required for analysis of the images, which is the case in Amsterdam where Mark works.

From the general public survey, we can extract that an ultrasound measurement was highly preferred over endoscopy by both the general public and IBD patients. Furthermore, we received useful information from the IBD patient interviews regarding the experience with endoscopy and the preparation for the procedure. The main issue in the preparation for the endoscopy procedure is the use of laxatives. The ultrasound measurements do not require the intake of laxatives and fasting. This is seen as a huge improvement by IBD patients. More on this can be found in the Implementation. Furthermore, the GMO acceptance among the IBD patients was very high as all of the interviewed patients would consider the intake of GMOs for diagnostic procedures.

With this confirmation in mind, we approached three gastroenterologists in the Netherlands. It soon became clear that our vision, a non-invasive, specific, fast-imaging, cost-effective, and easy-to-use diagnostic method for IBD by use of ultrasound, was not realistic as the current endoscopy is the golden standard and is difficult to improve. However, the monitoring of IBD, i.e. the POC fecal calprotectin home test (IBDoc) together with the periodic survey, can certainly be improved. Marieke, Mark, and Maurice told us that the fecal calprotectin test lacks specificity, which often leads to unnecessary follow-up endoscopies. Furthermore, no spatial information can be extracted from this test.

It was therefore decided to switch our goal from IBD diagnosis towards IBD monitoring. This is the point that we envisioned IBDetection; a non-invasive, specific, fast-imaging, cost-effective, and easy-to-use monitoring method for IBD by use of ultrasound. Here, we prioritize specificity and non-invasiveness because these shortcomings were most prominently emphasized in discussions with experts in the field of IBD. IBDetection gives a quantitative measure of the seriousness of the disease and locational data can be obtained. Also, IBD therapy response can be monitored in this way. A positive therapy response corresponds to a decrease in gas vesicle formation.

Also, Mark's other suggestion, improving the distinction between IBD and IBS, has been considered. This distinction is currently done by use of the fecal calprotectin test as well. Maurice indicated that this test, however, is accurate enough, and easy and quick, for the distinction between IBD and IBS. Therefore, the fecal calprotectin test outweighed our technique and thus we did not dive deeper into this.

Interestingly, ultrasound measurements are already used in some clinics for the diagnosis of IBD. This, however, is not performed in every hospital because analysis of these ultrasound images requires trained operators. Furthermore, the acquired data, e.g. the intestinal wall diameter, only indicates whether or not inflammation is present. Therefore, it is essential to clarify the added value of our technique. On Implementation, we further expand on the localization and quantification of disease severity.

Application

Now that our goals had been established, we set out to develop our Proof of Concept by performing in vitro lab experiments and building a computational model to simulate and test our system. Along the road, we faced several challenges and had to closely collaborate with experts in the fields of synthetic biology, ultrasound technology, and modeling. The most decisive discussions and subsequent integration are summarized below.

Human Practices

Integration

MSc. Bente Somsen - Synthetic biology expert

Bente Somsen is a PhD candidate at the Eindhoven University of Technology (TU/e) in the group Chemical Biology of the department Biomedical Engineering and is experienced with the use of various types of E. coli bacteria and (co-)transformations. We contacted Bente for advice about which cell type is the most appropriate for our proof-of-concept. Furthermore, we asked Bente about important aspects that had to be taken into account while designing our plasmids and possible solutions to the corresponding problem with the origin of replication, encompassing two origins of replication that are part of the same incompatibility group.

The two mechanisms, explained in The Science, were tested in E. coli BL21 (DE3) and Nissle 1917 cells. We asked Bente which cell type we could use the best. She told us that there are various types of market available E. coli cells that have been optimized for either protein expression or DNA amplification. She supported our plan of performing the experiments for the expression of proteins in BL21 (DE3) and advised us to amplify the plasmids into XL10-Gold or Novablue cells.

Additionally, we constructed our first plasmid design consisting of three different plasmids from the two explained mechanisms and asked Bente to reflect on this plasmid design. She supported the use of different antibiotics for each plasmid. Moreover, she advised us to make use of plasmids containing controlled and induced protein expression, instead of continuous and constitutive expression. Furthermore, we asked for possible solutions to the problem with the origin of replication. Bente confirmed our thoughts that the same origins of replications may lead to suboptimal control over the plasmid and subsequently protein concentrations. Nevertheless, Bente mentioned that these suboptimal concentrations are often still sufficient for proof-of-concept. Therefore, she suggested co-transforming our three plasmids at once and testing if the plasmids work together. If the plasmids are not functional together, we could still construct a new design that only consists of two plasmids. In addition to all her advice, Bente provided several protocols to perform the experiments needed during our projects, including the transformation protocols of XL 10-Gold and Novablue cells.

Dr. Yan Ni - Synthetic biology expert

Yan Ni is a Postdoc at the TU/e in the research group Chemical Biology of the department of Biomedical Engineering and is experienced in identifying and researching recombinant proteins. She is an expert with GMOs and plasmid transformation, which was really useful throughout the entire project. To help us with our research, weekly meetings with her were scheduled, in which we could ask all questions regarding the lab work, e.g. the problem with the origin of replication as described in the meeting with Bente. In addition, she supervised us inside the lab and was always available for smaller questions, such as machine settings. The three most influential aspects discussed during the meetings with Yan are: troubleshooting about our unsuccessful transformation, discussions about protocols, and the execution of the experiments, such as PCR and SDS-PAGE. To improve the protocol of the transformation, she suggested switching culture growth media from LB to SOC, to increase the survival rate of the bacteria. In addition, she came up with the idea of transforming a third plasmid into a bacteria which already contains two plasmids, to further increase the success rate of the transformation. For the execution of the experiments, she explained how to measure the fluorescence of mCherry, GFP and mRFP1 in a plate reader and spectrophotometer.

Dr. Alexander Gräwe - Synthetic biology expert

During our time in the lab, Alexander Gräwe was very interested in our project, as he was part of the iGEM Bielefeld-CeBiTec Team of 2015. At the moment, Alexander Gräwe is a Postdoc of the Biomedical Engineering department in the research group Protein Engineering at the TU/e. He offered a lot of help throughout the project by providing us with his protocols and giving us key insides into the processes in these protocols. For example, he helped us with the expression of the TtrR protein, which is controlled by doxycycline. He supplied us with various tips to explore the reason why the TtrS/R sensing part might work without doxycycline. Moreover, we discussed various steps in the restriction and ligation protocols. In particular, the time of our ligation step, namely 30 minutes at room temperature. Alexander suggested a longer ligation time and a lower incubation temperature. Furthermore, he helped us in the lab with several steps considering these protocols. In addition, we discussed problems in the lab regarding part improvement, sequencing troubles, our unsuccessful transformation, and the problem with the same origin of replication as mentioned also in the meeting with Bente. Alexander suggested making use of self-made competent cells for both Proof of Concept and Part Improvement.

Dr. ir. Hans-Martin Schwab - Ultrasound expert

Through our limited theoretical knowledge and lack of practical experience with ultrasound equipment, we reached out to Hans-Martin Schwab. Hans-Martin Schwab is an assistant professor of the Biomedical Engineering department of Cardiovascular Biomechanics at the TU/e. We had multiple meetings with him and asked questions about the ultrasound image acquisition and the setup of the ultrasound equipment. Most importantly, we told him that we were working on a script to image our gas vesicles, the imaging script, and a script that can collapse our gas vesicles, the collapse script . Upon our explanation, he told us that we should make an image before and after the collapse of the gas vesicles at exactly the same spot with the same settings. These images can then be analyzed to visualize the concentration of the gas vesicles. Hans-Martin provided us with an already existing script for basic ultrasound imaging and helped us with making this script suitable for our specific experiment. In addition, Hans-Martin gave us information about the experimental setup.

MSc. Hein de Hoop - Ultrasound expert

For expanding the ultrasound assistance, Hans-Martin brought us in contact with Hein de Hoop to ask for further advice about the ultrasound scripts and setup. Hein de Hoop is a PhD candidate at the TU/e research group Cardiovascular Biomechanics of the department of Biomedical Engineering and the Catharina Hospital Eindhoven. Throughout the project we had multiple meetings, he explained how the already existing scripts for the ultrasound measurements work and he helped us with adapting the script to make our own imaging and collapse script, which corresponds to the experiments performed in the research papers [1,2]. After showing our first results of the ultrasound images, he gave the suggestion to angle the transducer, to reduce the reflection on the ultrasound images, since the images showed too much reflection. He explained to us why an angled transducer will reduce the reflection, and helped us with some of the settings of the script.

Prof. dr. ir. Tom de Greef - Modeling expert

Due to our limited knowledge of computational modeling, we were in close contact with our supervisor Tom de Greef for guidance on making the model. We had multiple meetings with Tom to discuss how to approach the modeling part of our project and to solve problems we stumbled upon during our design process. In our first meeting, he advised us to use the Matlab add-on Simbiology and provided us with some resources that we could use to get to know more about computational modeling. He also pointed out some important aspects we had to keep in mind while building the model, such as the units of the rate constants that were used, and validating each model version before moving on to a more complex version. During our meetings in a later stage of the project, he helped us out with achieving a realistic simulation, as we initially did not have a vision of what normal protein expression may look like in a bacterial cell. Therefore, Tom de Greef checked whether the time scale and concentrations in our simulation made sense for what we were trying to model.

By combining the information gathered by the multiple synthetic biology experts mentioned, we have adapted our project in a responsive way. First, upon the advice of Yan and Bente we made two plasmid designs for the integration of the TtrS/R sensing and the ARG reporter part. Second, we used plasmids with a controlled and induced protein expression. Third, we performed expression-based experiments into BL21 (DE3) and TOP10 cells and performed DNA amplification in Novablue, XL10-Gold, and TOP10 cells as this was advised by Bente and Alexander. Fourth, we adjusted the restriction and ligation protocols according to the advice of Alexander. Fifth, we changed our growth media to SOC, to increase the survival rate of the bacteria. Last, we used several protocols received from the experts. Read more detail of all the aspects discussed with these experts in Proof of Concept and Notebook.

Through the meetings with Hans-Martin and Hein we could perform the ultrasound experiment by ourselves, which made us more flexible to perform the experiments. Furthermore, we integrated a lot of tips about the scripts, which made it possible to optimize the visualization of the gas vesicles. The advice of Hans-Martin and Hein is explained in more detail on our Engineering Success page segment Ultrasound. Finally, we executed the advice about angling the transducer to reduce the reflection at the bottom on the ultrasound images, and it worked!

Upon Tom’s advice, we used the Matlab add-on Simbiology to build our model. However, this didn’t go without trial and error. So when we got stuck on our model, for instance because an error occurred that couldn’t be solved by using our normal strategy, a meeting with Tom was usually planned to take a look at the error together. Tom then provided us with strategies that we could apply to make our model more solid. These strategies included revising the rate constants and their units to make them more coherent across the whole model, and rebuilding the transcription and translation part of the model. We implemented both of these tips, and eventually were able to solve the errors.

More on Tom his guidance, and how we implemented his advice in our project can be found under Engineering Success.

References

1. Bourdeau, R. W., Lee-Gosselin, A., Lakshmanan, A., Farhadi, A., Kumar, S. R., Nety, S. P., & Shapiro, M. G. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature, 553(7686), 86–90. https://doi.org/10.1038/nature25021

2. Lakshmanan A, Lu GJ, Farhadi A, Nety SP, Kunth M, Lee-Gosselin A, et al. Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI. Nat Protoc. 2017;12(10):2050–80.

The Pill

Eventually, the GMOs would be implemented in a pill that can be ingested orally by the patient for IBD monitoring. For the design of the pill, multiple factors have been taken into account, e.g. the coating of the pill and the target area. We talked to multiple experts to set out the intricacies of our pill design and created IBDetection.

Human Practices

Integration

Mark Lowenberg - Gastroenterologist

For the design of our pill, we wanted to know at what position in the gastrointestinal (GI) tract our GMOs need to detect the inflammation. From literature, we learned that Crohn’s disease affects the entire GI tract and Ulcerative Colitis is confined to the colon making the release profile hard to determine. Therefore, we asked Mark Lowenberg where, in his experience, inflammation happens the most in the intestine. He told us that Crohn’s disease predominantly is present in the terminal ileum and colon and Ulcerative Colitis is indeed confined to the colon.

Bruno Pot - Microbiologist

Bruno Pot is a microbiologist who works for the company Yakult Europe in the Netherlands. He has researched the effects of probiotics on IBD and has experience with the delivery of bacteria to the intestines. During the meeting with Bruno, we wanted to establish the choice of bacteria for IBDetection. We identified that the initial choice to deliver E. coli Nissle 1917 bacteria to the intestines was not as obvious as we thought. Even though E. coli Nissle cells have been proved to survive well in the colon and have proven health benefits, Bruno explained that some E. coli bacteria have pro-inflammatory characteristics. Therefore, they have been associated with triggering flare-ups in IBD [1]. In addition, studies have revealed that there might be a connection between the administration of E. coli Nissle bacteria and an increased risk for colon cancer [2,3]. Bruno advised us to take a good look at the risks of E. coli bacteria, and possibly switch to a different organism. He suggested considering gram positive lactic acid bacteria, as they are physiologically resistant to low pH environments as found in the GI tract and already present in the gut microbiome. Examples for which most information is available are Lactococcus lactis or Lactobacillus plantarum, now named Lactiplantibacillus plantarum [4].

Gastroenterologists Catharina hospital Eindhoven

We got the opportunity to present our work for the gastroenterologist of the Catharina hospital in Eindhoven. During the discussion, we asked them several questions concerning the design of the pill. First, we had to decide if we should release the GMOs in the intestine or anchor them inside the pill. Anchoring the bacteria in the pill, however, requires sufficient resident time in the intestine to facilitate gas vesicle production. The gastroenterologists told us that a normal pill would reside for approximately 4 hours in the small intestine. Second, we asked the gastroenterologists if information regarding the localization of the inflammation is an important factor to take into account. They told us that the location is indeed important to know considering drug selection. Third, we were wondering if released GMOs were able to influence the already disturbed microbiome of IBD patients. They ensured us that it is very unlikely to disturb the microbiome, and especially with the Lactobacillus plantarum strain.

Alexander Willemse and Paul van Zutphen - BioConnection

BioConnection provides companies with development and manufacturing capabilities to bring the product from idea to the clinic. They helped us with the design of the pill, and in particular with the release profile of the GMOs. They sent us an article in which different medical release systems for use in IBD patients were elaborated.

Article - Oral Drug Delivery for Regional Targeting in the Gastrointestinal Tract [5]

The oral route for drug delivery is the most commonly used route for drug administration in the GI tract. However, this oral route can be challenging as the GI tract is complex and displays several physiological barriers that affect drug delivery. The physiological factors that influence drug delivery are; 1) gastrointestinal transit time, 2) gastrointestinal pH, 3) gastrointestinal mucus, 4) intestinal fluid volume, and 5) gastrointestinal enzymes, and 6) microbiome. The physiology of the GI tract in IBD patients is affected in multiple other ways, including increased mucus production, disrupted intestinal barrier due to mucosal surface alterations and ulcers, and the infiltration of immune cells. Therefore, targeting the GI tract in IBD patients is considerably different and more challenging compared to a “healthy” GI tract.

The paper describes three ways on which release can be based; 1) pH-responsive dosage forms, 2) time-dependent dosage forms, and 3) biodegradable dosage forms. These three factors, however, are considerably altered during an IBD flare-up. Diarrhea affects the intestinal volume, transit time, and mucosal integrity. This, in turn, can alter microbial metabolism and the secretion of enzymes. Furthermore, IBD patients have a more acidic colonic pH that largely varies in time. Thus, active inflammation significantly alters the physiology of the GI tract making conventional oral drug delivery approaches useless. Therefore, combinations of these colon-targeting strategies have been used in formulations for IBD patients. The combination of pH-responsive and time-dependent strategies is commonly used for drug delivery to the colon. Pulsincap® and Entocort® are both formulations based on this combination.

Alexander Willemse and Paul van Zutphen - BioConnection

At the end stage of our project, we went to Oss where BioConnection is located. We asked Alexander Willemse and Paul van Zutphen the final questions related to our pill design. First, we asked them how we could keep the GMOs alive inside the pill. They told us that the GMOs should be placed in a medium where sufficient amounts of nutrients are available. The amount of nutrients determines the shelf life of the pill. Another option was to put the GMOs in a dormant state in the pill. Upon a trigger, e.g. body temperature, the GMOs are again activated after intake. Additionally, we asked them to elaborate on the manufacturing of pharmaceutical products. They emphasized that pharmaceutical products have to be maintained at high standards to ensure the quality and purity of the final product as well as the strength of the active ingredients. Controls and checks are continuously executed to ensure medicines to be at a high level of precision and safety for every new produced batch. Therefore, it is essential to implement the industry-accepted Good Manufacturing Practices to maintain efficacy and safety of the pharmaceutical products. Furthermore, we wanted to have an indication of how much our pill would eventually cost when the production process is fully automated. This was really hard for them to answer as many things should be taken into account. Their rough guess was that a 10 Liter solution of GMOs costs about 10.000 euros.

With the help of multiple experts, we were able to come up with a design for our pill. First, we determined the target location. With Mark's help, we set the terminal ileum and colon as the target location of IBDetection, as this is the place where inflammation is mainly present. Second, Bruno helped us to determine the delicate choice of bacteria. E. coli Nissle 1917 as a delivery vehicle may have unforeseen downsides which forced us to consider switching bacterial hosts. Therefore, we decided, upon the advice of Bruno Pot, to use a selected lactic acid bacteria for future research, preferably a strain that is already used for genetic research, has probiotic properties, and is present in the human gut microbiome. A good candidate would be strain L. plantarum WCFS1 [6,7]. Third, the gastroenterologist of the Catharina hospital Eindhoven helped us to decide whether or not we should release our GMOs at the target location. From the model, we learned that 25 hours after induction maximum gas vesicle formation was reached. Therefore, anchoring bacteria inside the pill, which only stays for 4 hours in the small intestine, was not an option. For our pill design, we thus decided to release the bacteria in the intestine as the distortion of the microbiome was highly unlikely. Furthermore, the full release also facilitates the extraction of the inflammation localization, which is an important parameter.

With BioConnection we discussed the coating, shelf life, manufacturing, and costs of the pill. They provided us with an article in which different release strategies for IBD medications were discussed. From this article, we learned that a combination of strategies is often used for the targeting of the intestine in IBD. Therefore, we decided to use a pH-sensitive and time-dependent coating for our pill design. In addition, they told us that the shelf life of IBDetection is fully dependent on the number of nutrients available in the pill. This is important to take into account while producing the pill. Another option is to keep the GMOs in a dormant state that is activated upon a trigger, e.g. body temperature. In this way, fewer nutrients are required. In addition, for the manufacturing of IBDetection, it is essential to implement the industry-accepted Good Manufacturing Practices to maintain safety and efficacy of IBDetection. Furthermore, the rough estimation of 10.000 euros per 10 Liter of produced GMOs were used to determine the costs of a single pill. One pill will contain approximately 2 mL of GMOs which results in a total cost per pill of 2 euros. The final pill design is visualized on Implementation.

References

1. Palmela C, Chevarin C, Xu Z, Torres J, Sevrin G, Hirten R, et al. Adherent-invasive Escherichia coli in inflammatory bowel disease. Gut [Internet]. 2018 Mar [cited 2021 Oct 18];67(3). Available from: https://pubmed.ncbi.nlm.nih.gov/29141957/

2. Nougayrède J-P, Chagneau CV, Motta J-P, Bossuet-Greif N, Belloy M, Taieb F, et al. A Toxic Friend: Genotoxic and Mutagenic Activity of the Probiotic Strain Escherichia coli Nissle 1917. mSphere. 2021 Aug 25;6(4):e0062421.

3. Wassenaar TM. E. coli and colorectal cancer: a complex relationship that deserves a critical mindset. Crit Rev Microbiol. 2018 Sep;44(5):619–32.

4. Spath K, Heinl S, Grabherr R. “Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete.” Microb Cell Fact. 2012 Oct 25;11(1):1–8.

5. Hua S. Advances in Oral Drug Delivery for Regional Targeting in the Gastrointestinal Tract - Influence of Physiological, Pathophysiological and Pharmaceutical Factors. Front Pharmacol. 2020 Apr 28;11:524.

6. Mercenier A, Pavan S, Pot B. Probiotics as Biotherapeutic Agents: Present Knowledge and Future Prospects. Curr Pharm Des. 2003 Feb 1;9(2):175–91.

7. Bruno. Probiotics from research to market: the possibilities, risks and challenges [Internet]. Unknow; 2016 [cited 2021 Oct 18]. Available from: https://dokumen.tips/documents/probiotics-from-research-to-market-the-possibilities-risks-and-challenges.html

Entrepreneurship

The ultimate goal of IBDetection is to reach the market. For this, however, investors are crucial. Therefore, we reached out to Brabant Ventures (BOM), who helps ambitious Brabant companies to grow in a future-proof manner. Moreover, BOM supplies funding to startups and scaleups in Brabants' top industries including Life Sciences & Medtech.

Human Practices

Integration

Paul Vernooij - Investment analyst

Paul Vernooij works for BOM at the Life Sciences & Medtech department. We reached out to him for advice on how we can make our project more attractive for investors. During this meeting, Paul told us that having one or multiple patents on the product is very important. Furthermore, excluding risks in the preclinical phase, for example through toxicity studies, reduces capital risks for investors. This is because the multiple phases of clinical studies are extremely expensive. In addition, it is crucial to show the potency of the product. This can be done by presenting actual acquired data, e.g. pharmacokinetic or pharmacodynamic studies. Last but not least, dividing capital risk over multiple investor parties and having a good feeling about the team behind the product is crucial.

After the conversation with Paul, we were able to come up with a plan for our pill for the future. To reach the market, lots of money is required. Therefore, we need to make our pill interesting for investors. This can be achieved by doing the following things; 1) reducing risks in the preclinical phase, 2) apply for one or multiple patents, 3) show the potency of the pill, 4) have a close and driven team, and 5) have multiple parties that want to invest to divide capital risks. When these things are kept in mind, the pill has the highest chance to reach the market .

Ethics

The intake of GMOs is required for IBDetection. The GMO legislation, however, is very strict due to safety and ethical reasons. We spoke with iGEM team BOKU Vienna about the ethical concerns related to our pill design and came up with an alternative. Furthermore, together with them, we arranged a meeting with Johannes Rath to discuss the ethical concerns related to both our projects and to discuss how we can perform a patient interview in a responsible manner.

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Integration

iGEM team BOKU Vienna - Partner

iGEM team BOKU Vienna pointed out that the full release of the GMOs in the intestine comes with ethical concerns. Therefore, we elaborated upon the alternative options that can suit IBDetection. As they also aim to target the intestine with their project Friendzyme, their approach might be suitable for our pill. In short, they create a biocompatible capsule that has pores where enzymes go out and the GMOs stay in. This capsule is created out of a positively charged and a negatively charged polymer creating a polyelectrolyte complex. Furthermore, thiolated chitosan of the complex can bind cysteine in the intestinal mucosa by the use of disulfide bonds, resulting in a longer residence time.

Johannes Rath - Ethical expert

At the end of our project, we got the opportunity to discuss the ethical concerns related to IBDetection with dr. Johannes Rath. Johannes works as an ethics, biosafety, and biosecurity advisor for various international organizations including the European Union and the United Nations, making him clearly the right person to speak with. He directly told us that the field of GMOs in Europe is very complicated. This is mostly due to the three dimensions of safety; safety of research subjects, environmental safety, and horizontal gene transfer. Furthermore, safety and ethics are very closely related.

We asked Johannes to elaborate on the ethical issues related to the implementation of IBDetection on the market. He told us that approval must be granted by the competent authorities. Here, you have to show that your project is superior to the currently used method, being the threshold. Preclinical and clinical studies have to be performed to obtain output parameters. These output parameters must be significantly better than the threshold parameters, yielding more benefits than the current technique. The ethical question here is: "What is significantly better?". Furthermore, you have to demonstrate that the technique possesses no or a limited amount of risks. Johannes emphasized that you must see the risks independently of the benefits. Thus, pointing out that, the benefits outweigh the risks is not enough. The ethical question here is; "What risks are acceptable?". Both these questions are very difficult to answer. However, in the end, the competent authorities make the final call and determine whether a product gets approval.

Another ethical issue we discussed relates to responsible communication of our research to patients. By conducting patient interviews, we risked giving patients false hope for a better monitoring procedure. This is undesirable and in the end painful because the product might never reach the market. We asked Johannes how we could responsibly perform patient interviews. Johannes told us that this is a general problem that happens with many research topics. He told us that responsible communication is of high importance. To avoid giving false hope, he advised us to stress that we are at the beginning of the research and that it takes many years before it potentially reaches the market. Even if it does not reach the market, you contribute to the general knowledge and understanding of the disease, enabling future research. Johannes emphasized that self-testing happens more and more within the do-it-yourself biology circles, but runs outside the current ethical framework. It should be prevented as it might not provide suitable results and can have negative impacts on individual participants. Examples that highlight the negative consequences of applying unproven treatments exist [1].

iGEM team BOKU Vienna pointed out their ethical concerns related to our pill design. The release of GMOs is ethically irresponsible, and therefore they wanted to help us establish an alternative pill design. As they also aim to target the intestine, their approach can suit as an alternative for our current pill design. In this alternative design, thiolated chitosan in a polyelectrolyte complex can bind cysteine in the intestinal mucosa by the use of disulfide bonds, resulting in a sufficient residence time for gas vesicle formation. Using this approach for IBDetection comes with several challenges which will be touched upon on Implementation. However, as the GMOs stay inside the pill, the ethical concerns are significantly reduced.

Furthermore, Johannes told us that safety and ethics are very closely related. Performing a proper safety assessment is thus of high importance. Therefore, we looked in more detail at several Safety aspects related to IBDetection including the safety of research subjects, environmental safety, and horizontal gene transfer. Furthermore, Johannes told us that in order to get approval granted from a competent authority, preclinical and clinical studies must be performed. The output parameters must be significantly better than the fecal calprotectin test being the threshold. Moreover, the benefits of IBDetection must be seen independently from the associated risks. Associated ethical questions are 1) "What risks are acceptable?" and 2) "When are output parameters significantly better?". These questions are hard to answer, and the final call lies in the hands of the competent authority. Additionally, he advised to stress that we are at the beginning of the research and that it takes many years before it potentially reaches the market and is integrated into our patient interviews. In this way, we tried to avoid giving false hope and thus perform responsible interviews.

References

1. Abbott A. Austria reinstates disgraced doctor. Nature. 2011 Sep 20;477(7365):384.

GMO Legislation

From the meeting with the Dutch National Institute for Public Health and Environment about the Safety of IBDetection, we learned that the GMO legislation is very strict in Europe. To gain more information on this topic, we got the opportunity to talk to Lucie McMurtry from EuropaBio. During this meeting, we discussed the scope of the GMO legislation within the European Union, current developments, and future perspectives for our project in Europe.

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Lucie McMurtry - EuropaBio

Lucie McMurtry works as Industrial Biotechnology Officer at EuropaBio, which is Europe's largest and most influential biotech industry association. EuropaBio is committed to the socially responsible use of biotechnology to improve quality of life and to prevent, diagnose, treat, and cure diseases. Furthermore they aim to improve the quality and quantity of food and feedstuffs and move towards a biobased and zero-waste economy.

During our meeting, Lucie was kind enough to give a presentation about the topics that are important to us. For our project, two directives are of importance: Directive 2001/18/EC on the deliberate release of GMOs into the environment, and Directive 2009/41/EC on the contained use of genetically modified microorganisms (GMM) [1,2]. The first directive describes a prior authorization system for the deliberate release of GMOs, and differentiated rules for experimental release and placing on the market of GMOs. The second directive lays down rules for the contained use of GMOs, to protect human health and the environment in the EU. These rules are very strict, and as a consequence, there is currently no GMM on the market for deliberate release, as only contained use is considered feasible.

Lucie explained that GMO legislation is outdated and slow to change, and thus deliberate release will likely not be possible in the scope of our project. Even though there are many companies involved in EuropaBio that are interested in the deliberate release of GMMs, there are no clear paths for developing frameworks for deliberate release. Lucie mentions how the outdated legislation is frustrating for these companies, as it limits the potential of the technology heavily and could even potentially result in companies migrating out of the EU. Nonetheless, Lucie states that fundamental research like ours, on the potential and efficacy of using GMOs in the healthcare sector is paramount to the future development and adoption of GMO use in the biotech industry.

In terms of the stigma on GMOs, Lucie presented us with the results of a recent Eurobarometer survey on food safety in the EU. The results showed that most Europeans hardly care about GMOs, but that the most concern is about antibiotics, hormones, and steroid residues in food.

With the current EU legislation, it is very unlikely that IBDetection would reach the market in the near future. Even though there are few risks involved in the GMOs that we will use, the regulatory framework does not allow for the deliberate release of GMOs into the environment, which our pill requires. Furthermore, the concerns about GMOs are much lower compared to the concerns about antibiotics, hormones, and steroids residues under Europeans. For our project, this infers that we should strongly take into consideration how to avoid the resorption of material from our final product in the intestine to make the product more appealing to the user.

References

1. EUR-Lex - 32001L0018 - EN - EUR-Lex [Internet]. [cited 2021 Oct 18]. Available from: http://data.europa.eu/eli/dir/2001/18/oj/eng

2. EUR-Lex - 32009L0041 - EN - EUR-Lex [Internet]. [cited 2021 Oct 18]. Available from: http://data.europa.eu/eli/dir/2009/41/oj/eng