Mr. Shreyas Samir Parkhie introduced the concept of designing a targeted pill, leading us to create a colon-targeted pill.

Mr. Prameya advised us to narrow our focus to a specific subtype of IBS, which enhanced the efficiency of our treatment approach.

Ms.Shubhra Singhal recommended conducting an educational activity at the Regional Science Centre, a relevant audience circle in the Tirupati district. Additionally, she encouraged us to explore the availability of health insurance for IBS patients, recognizing that mental health-related diseases were often excluded from coverage.

Ms. Lochan Chaudhari Bionumbers and Salis lab introduced used for parameter hunt and estimation

Mr. Jhagan R advised us to investigate the storage conditions of probiotics. Consequently, we visited medical shops to gain insights into the ideal probiotic storage methods.

Ms. Manasa N S and Ms. Asma Shirin gave us the idea to start modelling the system with diffusion instead of facilitated transport. This made the modeling simpler and complexity was slowly added to the model later.

We reached out to Dr. Usha Madhuri to gain insights into IBS, its existing treatments, and diagnostic approaches. Dr. Madhuri explained that IBS is a common gastrointestinal disorder often linked to unhealthy dietary habits, including the consumption of hard-to-digest, spicy, and junk foods and disruptions in gut microbiota. The primary approach to managing IBS involves lifestyle modifications. Medications commonly used include antibiotics to eliminate pathogenic bacteria responsible for gut microbiota disruption and probiotics to aid in restoring a healthy bacterial balance.

Integration:

Incorporating this information into our work in synthetic biology, we were inspired to explore the development of a probiotic bacterial strain as a potential treatment for IBS.

We reached out to Dr. Avinash for a comprehensive overview of IBS, including its causes, symptoms, diagnostic methods, available treatments, and other related details. During his presentation, Dr. Avinash discussed the rising prevalence of IBS among patients over the years and provided detailed information on the following aspects:

• Scientific definition of IBS
• Subtypes of IBS
• Prevalence of IBS
• Symptoms of IBS
• Associated Comorbidities with IBS
• Pathophysiology of IBS 
• Diagnostic criteria, approaches and differential diagnosis
• IBS management
• Existing treatments for the different subtypes of IBS
• Emerging therapies for IBS

Link for presentation: https://static.igem.wiki/teams/4836/wiki/human-practices/irritable-bowel-syndrome-whats-new-in-management-1.pdf

Integration:

After a comprehensive introduction to IBS, including its various subtypes, we made the decision to target serotonin levels as a potential treatment approach for IBS. This decision was motivated by serotonin's varied role in the gut and brain. We specifically focused on the IBS-D subtype because IBS-D patients exhibited increased serotonin levels in the gut, and this subtype is particularly concerning due to the potential overlap of diarrhea symptoms with other diseases. Additionally, we received valuable advice to create an IBS-D Patient Survey. The reasoning behind this was that IBS symptoms can vary significantly from person to person, making it crucial to gather insights from different patients' perspectives. This survey would aid in developing our project to a genuinely beneficial level to society.

We conducted an interview with an IBS-D patient to gain insights into their perspective on Irritable Bowel Syndrome.

Here's a summary of our discussion:

Q: How do you define IBS, and how long have you been suffering from the disease?

A: “I've been dealing with IBS for around a decade now. It means experiencing uncontrollable urges to pass watery stools, particularly after consuming certain foods.”

Q: What do you believe are the causes of IBS?

A: “I believe that the extreme stress I faced in my personal and professional life played a significant role in developing my condition.”

Q: How often do you experience the need to pass stools?

A: “I typically have the urge to pass stools 2-3 times in the morning, almost daily. It can intensify when I consume triggering foods or become anxious, and this urge can persist for an extended period.”

Q: What are the triggers for your IBS symptoms?

A: “Foods are my primary triggers, especially fiber and protein-rich foods. Items like wheat, dairy products, most fish, and bananas are problematic. However, without issues, I can comfortably include rice, beans, small fish, tea, and carrots in my daily diet.”

Q: What treatments have you tried for your IBS?

A: “I've consulted various doctors, including physicians and gastroenterologists, but I've had limited success with prescribed medications. When my symptoms worsen, I usually take anti-diarrheal tablets, paracetamol, and gas-relief tablets like Gelusil. Additionally, I find relief through yoga and meditation, which have helped me manage my anxiety, an important aspect of my condition.”

Q: Do any of your family members also suffer from IBS?

A: “To the best of my knowledge, no one else in my family has IBS.”

Q: What are the typical symptoms you experience with IBS?

A: “My symptoms primarily involve the urgent need to pass stool, accompanied by bloating. Fortunately, there isn't much pain.”

Q: Do your symptoms worsen before or during your menstrual period?

A: “My symptoms don't appear to be related to my menstrual cycle.”

Q: How has IBS impacted your quality of life?

A: “IBS has significantly restricted my diet choices, making me hesitant to try new foods. The unpredictability of bowel movements has caused social anxiety and reluctance to go out in public. It has also disrupted my sleep patterns, as good sleep greatly affects my condition. On the flip side, triggers can disturb my sleep, and I resort to melatonin tablets to improve my sleep quality.”

Q: Are you comfortable discussing IBS openly?

A: “No, I'm typically hesitant to discuss my condition with friends or colleagues, as many people are unaware of it.”

Q: What improvements would you like to see in IBS healthcare?

A: “I'd like to see cause-based treatments rather than symptom management and accurate diagnostic techniques. Additionally, improved sleep management is essential.”

Q: How are you coping with your disease?

A: “I'm closely monitoring my lifestyle, trying to identify the causes of my symptoms and understand my condition better. This approach, coupled with a healthy and disciplined lifestyle, has been more effective for me than relying solely on medication.”

Integration:

It's clear from this conversation with the IBS-D patient that there is a pressing need for improved medications and a deeper understanding of the causes of the disease. Stress, anxiety, and diet emerged as major factors associated with IBS. This insight suggests that further research in these areas would be valuable for developing more effective treatments and management strategies for individuals with IBS. Addressing the root causes, such as stress and diet-related triggers, can significantly enhance the quality of life for those living with this condition.

https://www.researchgate.net/profile/Vineet-Ahuja-2

We conducted an extensive literature review on IBS and discovered that serotonin levels in the gut are elevated in both IBS-D (Irritable Bowel Syndrome with Diarrhea) and IBD (Inflammatory Bowel Disease). Furthermore, our research revealed a downregulation of serotonin transporter, which leads to the accumulation of serotonin in the gut. Intriguingly, the causes of IBS and IBD share significant similarities. As part of our effort to gain deeper insights, we reached out to the renowned gastroenterologist Dr. Vineet Ahuja. Our objective was to understand the role of serotonin and explore the potential benefits of regulating serotonin levels in the treatment of both IBS-D and IBD.

Discussion:

Q: How are serotonin levels relevant in IBS and IBD?

A: “As serotonin is a neurotransmitter, it affects the appetite sensation, gut motility and permeability. The causes of these diseases are unspecific, and thus, reducing serotonin levels alone cannot treat these diseases. It has a major role to play in the regulation of bowel movements, especially in IBS. Levels of serotonin have less role in IBD as immune response and inflammation are the main factors contributing to the disease, but serotonin has an effect in regulating the permeability of the gut as serotonin is a pro-inflammatory molecule. There are existing medications for IBS targeting serotonin receptor antagonists, which work on the nerves and smooth muscles of the GI tract.”

Q: What are the side effects associated with existing serotonin-targeted therapy?

A:

• Severe constipation and ischemic colitis
• Headaches (migraines),
• Dizziness
• Back or joint pains.
• Nausea/vomiting and diarrhoea
• Cardiovascular risk 

Q: Can a drug reducing serotonin level play a role in treating IBD and IBS?

A: “It would be beneficial for IBS treatment and can be given as a combination drug for treating IBD”.

Q: How common are probiotics used for the treatment of Gastrointestinal diseases?

A: “Gut microbiota plays a major role in the development and prevention of gastrointestinal diseases, and thus probiotics are widely prescribed. They are found to be very effective in the treatment of gut disorders and maintaining gut as well as mental health. Escherichia coli Nissle, Lactobacillus, Bifidobacterium and Saccharomyces boulardii are the commonly used probiotic strains”.

Q: What metabolites, if produced by bacteria, can improve gut function?

A: “Short-chain fatty acids, Polyphenols, curcumins and organosulfur have beneficial effects on gut health.”

Q: How can we prevent gastrointestinal diseases?

A:“ Healthy diet pattern, reducing the consumption of processed food, high sugar content food, avoiding carbonated drinks and alcohols, maintaining hygiene and steeping forward from sedentary lifestyle can prevent lifestyle-related gastrointestinal diseases to a large extent. Living a stress-free and happy life is also important to improve gut health.”

Integration:

We got confirmation to move forward and work on serotonin for treating IBS and using probiotics as a combination drug for IBD treatment. He also commented that our probiotics can avoid the side effects associated with IBS drugs and is non-invasive as compared to others. We decided to choose our host organism as E. coli 1917 Nissle.

https://www.nottingham.ac.uk/medicine/people/robin.spiller

We reached out to Dr. Robin Spiller, whose research focuses on the pathophysiology of functional GI diseases, particularly emphasising the role of infection, inflammation, and changes in serotonin metabolism in irritable bowel syndrome (IBS). During our interaction, we presented our project to seek his expert opinions.

Q: Can our engineered probiotic treat IBS-D?

A: “Serotonin has a role in IBS, and serotonin-targeted therapies are beneficial. High levels and activity of serotonin is found in IBS-D

 For your project to be successful, first confirm

• Serotonin is present in the gut lumen so that bacteria can take it up and convert it into melatonin. 

• Luminal serotonin plays a role in regulating bowel movement

Q: What are the reasons for increased gut motility in IBS?

A: “There are several types of reasons for increased gut motility and sensitivity, like immune activation by bacteria and increased activity of neurotransmitters like serotonin and hormones like motilin. They can also arise as side effects of certain drugs or food. Sensations from the gut are perceived by the stimulation of receptors present in epithelial cells. These receptors are usually activated by stretching of the gut wall cramping, or inflammation leading to increased gut motility.”

Q: What are the levels of serotonin in the gut?

A: “It is very difficult to measure serotonin levels in the gut. The electrodes are inserted into mucosa to measure the levels of serotonin. Since platelets contain serotonin storage vesicles, biopsy-based measured serotonin levels are not accurate as platelets can break while taking the sample. Normal release by stimulation ranges from 1- 1.2 micromolars.”

Q: What is the role of serotonin in diarrhoea?

A: “Serotonin in gut has motor and sensory functions through submucosal and mesenteric neurons that respond to serotonin through receptors present on the cell surface. Serotonin activates the intrinsic and extrinsic afferent neurons in the enteric nervous system. serotonin is responsible for the induction of slow excitatory postsynaptic potentials (EPSP) recorded in enteric neurons. This activity being stimulated can lead to diarrhoea.”

Integration:

We clarified that the majority of serotonin produced is released into the lumen. Luminal serotonin has a role in serotonin activity as serotonin receptors 5HT1- 5HT7 are distributed uniformly across the epithelial cells.

https://www.iisertirupati.ac.in/faculty-details/?faculty_id=Bio-17&id=2124

Dr. Hussian Bhukya, the Principal Investigator for the iGEM 2022 team, played a crucial role in our project. We reached out to him to validate our wet lab plan, experimental procedures, and the techniques we intended to employ.

Our wet lab experiments comprised seven modules that required gene assembly. Dr. Hussian recommended the use of the infusion cloning method, citing its advantages as a scarless assembly technique and its relative ease of use. However, given that infusion cloning is most efficient for up to two inserts, and our project involved more than two inserts, we ultimately decided to opt for the Golden Gate assembly method.

Jeffrey, who battles IBS and IBD, is the founder of the IBS Support Group, which boasts a membership of 12,371 individuals. His website serves as a comprehensive resource, containing crucial scientific information about these diseases and their management methods. We intended to connect with Jeffrey to gain insights into IBS, engage with our stakeholders, and gain a deeper understanding of the various factors associated with the disease.

Discussion:

Q: How would you define IBS, and which subtype were you suffering from?

A: “IBS is a gastrointestinal disease that affects your lower abdomen, mainly small intestine and colon. The symptoms include abdominal pain, bloating due to gas formation and change in bowel habits, either constipation or diarrhoea or both. IBS is classified into subtypes IBS-D (Diarrhoea predominant type), IBS-C (Constipation predominant type), IBS-M ( both Diarrhoea and constipation or mixed type) and IBS-U ( Unsubtyped). PI-IBS (Post Infectious IBS). This classification is better for understanding the disease, but generally, symptoms are mixed, and thus, we cannot say that we belong to a particular subtype. Mostly, it was IBS-D for me. The specific causes of the disease are unknown. It is mainly associated with diet, accompanying an uncontrollable tendency to pass stools frequently. In the Mornings, I have a high frequency of tendency to pass stools.”

Q: How can IBS-D symptoms vary from person to person?

A: “Frequency, severity, and overall symptoms vary. Some patients can have diarrhoea without abdominal pain or bloating. Some suffer from severe diarrhoea, which would be manageable for others. But there would always be a trigger that leads to their symptoms. This can be due to their lifestyle, mental, and physical health.”

Q: How is IBS being diagnosed, and how can we differentiate its subtypes?

A: “IBS is very difficult to diagnose. The symptoms are very similar to many other diseases like Inflammatory Bowel Diseases. Rome criteria are currently used. Bristol’s stool chart is used to analyse stool type to prescribe medications. It ranges from 1-7.

 Type 1-2 indicate constipation,

 Type 3-4 are ideal stools as they are easier to pass, and

 Type 5-7 may indicate diarrhoea and urgency.

The application of these techniques is rare, and IBS has a high possibility of being misdiagnosed.”

Q: Are existing medications satisfactory in treating the disease?

A: “The existing medications are symptom-based, like anti-diarrhoeal, laxatives, anticholinergic, anti-depressants and painkillers. Probiotics are also being given to supplement these medications. They are unspecific and are associated with side effects like nausea, sexual side effects, headache and cardiovascular risks.”

Q: Can serotonin-targeted therapy help decrease the severity of IBS?

A: “Yes, serotonin was the target for the first drug for IBS, which targeted SERT. Serotonin regulates bowel movements in IBS patients.”

Q: Is melatonin good for gut health?

A: “I am a consumer of melatonin tablets, and melatonin is taken for better sleep. Its association with the gut is still unknown to me. There are studies that relate IBS and melatonin. You can refer to those for more information.”

Q: To what extent do lifestyle modifications manage IBS?

A: “Avoiding IBS triggers can help in managing IBS to an extent. High FODMAP diets are found to trigger IBS symptoms. These involve a sugar-intensive diet, a fibre-rich diet, dairy, processed food, alcohol consumption, caffeine and carbonated drinks. Stress and anxiety are something the majority of IBS go through. Cognitive Behaviour Therapy is found very useful for managing post-IBS stress. This reduces the social anxiety associated with IBS.”

Q: What inspired you to start an IBS Support Group?

A: “I suffered from IBS from a very young age, and I find it difficult to approach the disease. It is generally mistreated and ignored, and there is much irrelevant information widespread regarding IBS. There was a need to spread awareness about disease and help patients suffering from the disease. I thus decided to start a support group so that it wouldn't be difficult for sufferers anymore. We declared April 19 as World IBS Day. Tuesday Night IBS is a show intended to create IBS awareness, spread scientific evidence-based information, and support patients.”

Q: Is there medical insurance available for IBS Patients?

A: “No, IBS medications can be very expensive. Medications should be taken continuously and are very expensive. No medical insurance is available for IBS treatments. This can vary in different parts of the world.”

Q: What is the most difficult aspect of being an IBS Patient?

A: “In many patients, IBS is accompanied by social anxiety, hesitation towards travelling and underconfidence. Constantly being feared of experiencing uncontrollable bowel movements.”

Q: Are people open to talking about IBS?

A: “No, patients are generally reluctant to share about the disease or their symptoms openly. This can reduce the possibility of identifying the disease as well as its management. Many go undiagnosed, and it decreases their quality of life very drastically. There were many IBS awareness sessions and webinars conducted to remove the associated stigma related to IBS.”

Integration:

The IBS patient survey was prepared and sent through the IBS Support Group. We conducted a survey to test IBS public awareness and to educate people about IBS. We also conducted an Educational activity in the Regional Science Centre and distributed pamphlets for IBS awareness.

https://www.nvhealth.co.uk/

The survey analysis and patient interview suggest that diet management can play a very big role in treating IBS. We contacted Nicola Vaughan, a nutritionist to several IBS patients, to understand the relationship between diet and IBS, thereby acknowledge the scientific reasons behind diet management.

Q: Which are the foods that trigger or worsen IBS-D symptoms ?

A: “The triggers are mostly individual-specific, subtype-specific and vary from person to person. Generally, foods are classified as High FODMAPs and Low FODMAPs. The high FODMAP-containing foods are found to be the major triggers of IBS. The common triggers among common diets are histamine, high sugar and fibre-containing foods, dairy and oil-rich food, wheat, caffeine and alcohol. Wheat and gluten contain high levels of lectin, making them to trigger IBS symptoms.”

Q: Why are IBS Patients more sensitive to certain foods considering their normal effect on a person unaffected by IBS?

A: “The major cause of IBS is the disruption of gut microbiota. The variation of gut microbial composition among different people is majorly responsible for the specificity of triggers to an extent. Certain foods are associated with the growth of pathogenic bacteria, like high sugar content diet and fibre-rich food. Oils are inflammatory, and since immunity varies within the population, this leads to specific triggers. Certain foods like polysaccharides lead to water retention and gas production, thereby leading to bloating, pain and increased visceral hypersensitivity. Some individuals with IBS may have impaired histamine metabolism or increased sensitivity to histamine, leading to symptoms such as headaches, skin rashes, flushing, and gastrointestinal issues. The low histamine diet involves avoiding histamine-rich foods and histamine-releasing foods

Q: What is the suitable diet advisable for IBS patients?

A: “The ideal diet for IBS Patients is not low FODMAPs; there’s no such diet. It’s the elimination diet method that is suggested. This is the main technique that I suggest to patients. Try to avoid the suspicious food that you think will trigger your symptoms from your diet and see how it goes. Similarly, find your triggers, avoid them and identify your healthy diet. The low FODMAP diet is primarily focused on reducing the consumption of certain carbohydrates that are poorly absorbed in the small intestine. FODMAPs (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) can be fermented by gut bacteria, leading to gas production and symptoms like bloating, abdominal pain, and altered bowel movements. ”

Q: What are the foods that are good for mental health?

A: “Fat-rich food like olive oil, rapeseed oil, nuts, seeds, oily fish, avocados, milk and eggs. Vitamins and minerals are found in fruits and vegetables. Those rich in antioxidants, omega 3, especially Vitamin B and beta carotene, can improve your mental health. Tryptophan amino acid is essential for the production of neurotransmitters like dopamine and serotonin. Dark chocolates thus increase your mood. Probiotics are also found to improve your mental health, as bacterial production of short-chain fatty acids is essential for serotonin production. junk food, which has a negative role in building synaptic connections, thereby reducing learning and memory power.”

Q: What are the foods that disrupt gut healthy microbiota?

A: “High sugar-containing and fibre-rich food can increase the growth of pathogenic bacteria in the gut, reducing healthy gut bacteria growth. The healthy bacterial species include Bifidobacterium and Faecalibacterium, which help in improving immunity. Their undergrowth can result in reduced immunity. Lactobacillus species are found beneficial in forming a biofilm that inhibits the growth of other bacteria, thus preventing pathogens. IBS patients are found to lack important species like Lactobacillus and Bifidobacterium, which implies that either their disruption led to IBS or, due to IBS, they were destroyed, which leads to further disruption of gut microbiome balance. Spicy food can also disrupt gut microbiota as it produces flares and inflammation, producing an immune response. Antibiotic consumption can greatly affect gut microbiota composition”

Q: What are the foods that improve gut health and prevent gastrointestinal diseases?

A: “Probiotics are found to be very healthy in the gut as the bacteria help to build, repair and strengthen the intestinal barrier. Vegan foods are generally suggested to be good for the gut, but they are associated with high fibre content and are difficult to digest. Conversion from butter and ghee for cooking can improve gut health due to their anti-inflammatory properties. Avoiding alcohol, and caffeine, as well as refined grains and sugar, which can cause oxidative stress and acid formation, is good for health. Monash University is the best reference for understanding nutritional aspects”

Integration:

Our initial plan was to create an IBS handbook that would contain valuable information regarding appropriate dietary habits to raise awareness about IBS. Unfortunately, due to time constraints, we were unable to allocate sufficient time to complete the handbook. As an alternative, we produced pamphlets using the available information to disseminate awareness about IBS.

https://www.cedars-sinai.edu/research/labs/pimentel.html

Mark Pimental and his research team work on Irritable Bowel Syndrome, and he is the Executive director of the MAST program. The areas of greatest interest and work in the MAST Program include motility disorders of the GI tract, SIBO, IBS, metabolic diseases (such as obesity and diabetes) and many others, as they relate to the microbiome. He discovered the first blood test for IBS. They develop drugs, diagnostics and devices related to conditions of the microbiome. We presented our idea to him to understand it’s feasibility as well as to seek suggestions and insights for improvements.

Dr Mark Pimental mentioned that our project idea relies on strong scientific evidence, as serotonin plays a very big role in regulating gut motility and permeability. Thus, it has a very good potential to be a successful therapeutic if executed properly. Important points he mentioned:

•  About 60 % of IBS patients suffer from disruption of gut microbiota, and IBS was induced in a mouse model by injecting bacterial cytolethal-distending toxin B (CdtB).

•  The Biosafety of the product should be more advanced. Gut-based bacteria can also colonise the urinary tract, causing infection. He suggested us to use an MIT-developed co-factor-based or CRISPER-based kill switch.

• Ecoli, Klebsiella and Salmonella are known to be bad for gut health and are found pathogenic. Their growth can lead to increased gas formation, resulting in a bloating sensation.

• Low-grade inflammation is found in IBS caused by cytokine activation and immune response.

Integration:

We find an alternative for Ecoli 1917 Nissle in consideration as they have a consistent colonisation pattern and increased growth that might worsen IBS symptoms. Bacteroides fragilis is a gram-negative commensal, and they are found to reduce inflammation associated with IBD. We propose to employ genetically stable CRISPR-based kill switches in our bacteria for biosafety.

https://www.rgcb.res.in/harikumar

We contacted Dr. Harikumar to understand the biosafety concerns associated with our project.

As we are developing genetically modified probiotics, we would like to understand the legal biosafety measures and steps we have to undergo.

Q: What are the possible threats to biosafety in our project?

A: “At this stage, the project has very minute biosafety concerns as you are working with a commonly used strain of bacteria Escherichia coli BL21 (Model organism) and Escherichia coli Nissle 1917 as the host organism. All genetically modified organisms possess a minimal level of biosafety issues like horizontal gene transfer, abnormal colonisation and the consequences of offsite release. Interactions of the GMO with other bacterias in the gut is also a possible threat. A major issue associated with probiotics is environmental exposure.”

Q: What are the general rules to follow while creating a probiotic?

A: “Once the proof of concept stage is over, there are different biosafety levels in order to approve it as a consumable product. There are four biosafety levels for working with living organisms. This classification is based on the risks associated with different types of microbes. There are further animal trials and human testing. Further details of biosafety concerns can be found in the Guidelines for biosafety by the Department of Biotechnology in India.

Regulations & Guidelines for Recombinant DNA Research and Biocontainment, 2017

Integration:

We developed the following

Environmental Safety - Photoactive kill switch,

Off-site release - Colon targeted pill

We also contacted a microbiologist, Dr. Brenda Anne Wilson, to understand the interactions of our bacteria in the gut and colonisation of our bacteria.

https://www.instem.res.in/faculty/praveen

Dr. Praveen Vemula works on innovations in drug/prodrug delivery using soft and hard hybrid biomaterials as ‘next-generation biomaterials’ in the field of translational research. He is also working on the treatment of IBD. We contacted him to understand colon-targeted drug delivery systems that are safe and suitable for delivering probiotics.

 We presented our idea, and he suggested us to use our drug as a combination therapeutic for IBS. He mentioned that the development of gastrointestinal disorders is mainly due to the change in diet patterns and unhealthy gut microbiota. He suggested that there is the existence of new ingestable microdevices containing pill designs

Integration:

Our colon-targeted pill is made of micrometre-sized polymeric containers, for the engineered delivery of probiotics, using spray-dried probiotic microorganisms. To achieve colon-specific delivery, micro containers are loaded with probiotic bacteria and sealed with an enzyme-sensitive coating. A genipin-cross-linked chitosan coating is developed that (i) is stable at gastric and intestinal pH, (ii) has tunable swelling, and (iii) is degradable by the colon-specific bacterial enzymes. The chitosan–genipin–coated micro containers. The genipin-cross-linked chitosan coating enables an effective local delivery in the cecum and colon without any premature release in the small intestine. 

https://asm.org/Biographies/Brenda-Anne-Wilson,-Ph-D

Brenda Anne Wilson is a renowned microbiologist. Her works include the study of bacterial toxins secreted by E coli. When we contacted her for insights into the characteristics of our host organism, E. coli, in the gut and to gain a deeper understanding of the bacterium's properties, she expressed enthusiasm for our project. She even expressed her willingness to consider publishing a paper in her capacity as an editor for a journal if our research yields successful results.

Q. Is Ecoli Nissle 1917 ( our host organism) good for gut health?

A: “Ecoli are bacteria that multiply fast in the gut, so they, as probiotics, can easily colonise the gut. The bacteria have reduced immune response compared to other probiotics, and they are known to be effective in controlling diarrhoea and for the treatment of Inflammatory gut diseases. They are good at secreting molecules that are beneficial for improving health, especially to strengthen and repair the intestinal barrier.”

Q. What is the mechanism of bacterial adhesion and colonisation in the gut?

A: “Ecoli shows a persistent colonisation pattern as compared to the transient effect seen in lactobacillus. This is useful in IBS as it is a lifelong disorder. The exact mechanism is not known, and are found to form a biofilm layer but not in specific conditions. They produce OMVs and bacteria, which also help bacteria to travel long distances and adhesion. The vesicles get diffused to mucin and reach epithelial cells.”

Q.What could be the possible interactions of our engineered Ecoli Nissle in the gut, and is it concerning?

A: “Ecoli Nissle can compete with especially pathogenic abacterial species in the gut, which is the reason they are beneficial. Ecoli Nissle produces anti-bacterial peptides that inhibit the growth of other E. coli species in the gut. Their biofilm formation can also inhibit the growth of other bacteria. However, these interactions do not have negative roles as the studies suggest, especially in this species. This can happen naturally as well”

Q.What is the expected growth curve of probiotics in the gut environment?

A: “The nutrition in the gut can be considered unlimited; therefore, the growth should be taken under the conditions of unlimited resources. However, growth and effect vary from person to person. This depends upon nutrition and existing gut microbiota. Probiotics are not effective in some people, but for the majority, it works ”

Q.How can we regulate bacterial populations in the gut?

A:“There are Immunoglobins that are found to decrease the adherence of bacteria to gut. In this way, use of immunoglobins can be used to regulate bacterial growth if modelled properly. Further mucin, which is present in the mucus layer, will also inhibit the adherence to host cell surfaces. This information can be utilised to develop techniques that help in creating transient colonisation in Ecoli and excretion of probiotics.”

Q.How can we control horizontal gene transfer?

A: “Ecoli bacteria can easily take up the genes and plasmid from the surrounding environment. So, the possibility of horizontal gene transfer is high. But this is not expected to cause very dangerous effects. Ecoli Nissle has been engineered earlier as well. As Ecoli Nissle is a commonly used strain, genetical engineering has very negative roles as virulence genes are absent and thus reduced risk. Genetic material chopping enzymes can be used as kill switches to ensure safety.”

Integration:

Dr Brenda validated our approaches and provided new areas to research like the usage of immunoglobulin to induce transient colonisation thus for ensuring the biosafety of our probiotics. Kill switches that can destroy the genetic material of bacteria are one among the proposed kill switches.

https://svimstpt.ap.nic.in/depts/surge/surgestaff.html

Our team made a visit to the Sri Venkateswara Institute of Medical Sciences (SVIMS) hospital, renowned for attracting patients from across India seeking accessible and high-quality healthcare services. Our primary objective during the visit was to gain insights into the prevalence and causative factors of Irritable Bowel Syndrome (IBS) in India and to gather statistics on the number of IBS patients and the diagnostic techniques employed to distinguish IBS from other medical conditions. We had the opportunity to discuss our research proposal with Dr. Venkatarami during our visit.

Q: How is IBS diagnosed and what differentiates the subtypes? 

A: “IBS is an exclusion disease, and if we find that the patient is not suffering from any other disease, it is considered IBS. Subtypes are decided upon the symptoms the patient suffers, either diarrhoea or constipation or both Rome criteria. The symptoms are similar to that of IBD symptoms include bloody stools, fever, extreme weight loss, and anaemia.” 

Q: What are the prevalent causes of IBS from the patient analysis?

A: “ Most prevalence is found in the adult population who suffers from stress and anxiety. Especially the people who lack a social circle, like parents whose kids are away for work. Youth from hectic academic stress and also among the patients.”

Q: Do you think a serotonin-targeted therapy can improve IBS symptoms?

A: “Serotonin is among the targets on which current therapies rely. It is involved in regulating gut movements, and thus, it should work. However, regulating serotonin alone won't be able to cure IBS. Rather, it can reduce the symptoms and severity.

Q: Is melatonin healthy for the gut? What concerns do high melatonin levels possess?

A: “The role of serotonin in the gut is quite unknown. It can induce sleep, and IBS patients are supplied with melatonin tablets for better sleep.”

Q: How does IBS symptom vary from person to person?

A: “The severity and frequency of diarrhoea, the triggers such as food or any particular emotion, abdominal pain severity and causes vary from person to person. This is why treatment for IBS is completely symptom-based. They are given antidepressants, antidiarrhoeals and painkillers for controlling symptoms .”

Q: Why is there a female prevalence of IBS?

A: “ The way males and females process emotions are different, and this can be one of the reasons for female prevalence.“

Q: What is the average number of patients visiting the hospital?

A: “5% of patients suffering from gastrointestinal diseases suffer from IBS generally. The patient number increased over the years.”

Q: Do you think IBS is left undiagnosed among the population?

A: “IBS is generally regarded as an exclusionary disease. It cannot be dangerous or lead to the death of anyone. There is a large possibility of people being undiagnosed or misdiagnosed. “

Q: Which part of the population is prone to IBS?

“IBS is prevalent among the general population ranging from 18-50 years. If you are undergoing chronic stress or have a family history of IBS, then you have more chances of having IBS in the future. It is prevalent among women.”

A: Are IBS patients prone to other diseases or vice-versa?

“It is generally the other way around. If the person is not suffering from any other diseases, then it's more likely to be IBS.”

Q: Are existing medications effective in the treatment of IBS?

A: “All existing medications are based on symptoms and are prescribed according to the severity of symptoms. Further, some probiotics are given to improve gut health and prevent the control of diarrhea. The patients can control their symptoms, but they come back; thus, more efficient treatments that are cause-based are essential.” 

Integration:

We recognized the significance of raising awareness about Irritable Bowel Syndrome (IBS), so we organized an informative IBS awareness session at the Regional Science Centre. During the session, we distributed pamphlets containing information about the condition and recommended preventive measures. In addition to this, we conducted a survey designed to gauge the general public's knowledge about IBS. The survey was carefully structured to gather responses and educate participants about IBS through the questions themselves, encouraging greater awareness about the condition among the respondents.

We presented our idea to Dr. Sarat during the visit to the SVIM hospital Neurology department to understand the possible side effects of the reduction of serotonin and the production of melatonin in the gut. 

Dr. Sarat mentioned that decreasing serotonin levels would not be hazardous as serotonin production occurs in the brain in different ways, and gut serotonin levels can vary according to 

diet consumption. Melatonin, as it passes the Blood Brain Barrier, can reach the brain and can induce sleep. Thus, the levels should be maintained so that the patient doesn't feel drowsy after

taking the probiotic. The melatonin shouldn't affect normal melatonin production levels in the gut or brain.

Integration:

The mathematical modelling is done to find out the CFU of bacteria that is required in such a way that melatonin production per second is at levels which is safe for our body.

https://drnairshospital.com/doctor/dr-v-balachandran/

We contacted Dr. Balachandran to understand the statistics of IBS Patients and the diagnostic methods employed to differentiate IBS from other diseases. Furthermore, to understand the effect of neurotransmitters serotonin and melatonin on overall health.

The main points he mentioned

⚫ IBS is considered as an exclusion disease that is diagnosed positively by ruling out the possibilities of every other disease.

⚫3% of total patients coming for treatment and 25% of patients suffering from gastrointestinal tract diseases are IBS patients.

⚫ Melatonin-containing probiotic pills are prescribed for IBS treatments, and there exist safe levels that are harmless to our body. Melatonin can induce sleep.

⚫Capsule Endoscopies, breath tests, colonoscopy and biopsies are used to differentiate IBS from other gastrointestinal diseases by checking the presence of immune cells, ulcers, blood, etc, from samples.

⚫ A test for IgE can be done to understand pathogenic gut microbiota dominance.

⚫Serotonin accumulation is found in Crohn’s disease, and serotonin syndrome.

Integration:

Probiotic dosage can be prescribed during the night due to the possibility of inducing sleep. The probiotic can be used as a combination drug to treat IBD.

https://www.jnu.ac.in/content/vikasyadav

We participated in the All India iGEM Meet, which took place at IISER Bhopal, and our presentation was evaluated by Dr. Vikas, who served as one of the judges. Dr. Vikas was genuinely impressed by our project and acknowledged its potential to bring substantial societal benefits, particularly due to the high prevalence of IBS. He generously offered his assistance by proposing to conduct animal trials for our project in his laboratory. Furthermore, he emphasized the significance of maintaining a balanced level of serotonin in the gut, especially as reduced serotonin levels can contribute to IBS-C, underlining the importance of our research in this context.

Integration:

We identified a regulation mechanism so that bacteria will only activate the genetic circuit for the conversion pathway of serotonin to melatonin in the presence of excess serotonin. This is using a serotonin sensor.

https://wyss.harvard.edu/team/core-faculty/george-church/

In our search for a serotonin sensor within bacteria that would trigger the genetic circuit exclusively in response to elevated serotonin concentrations, we conducted an extensive review of existing literature. This thorough investigation led us to identify three potential methods for detecting serotonin.

5HT1A Receptor in Ecoli:

Scientists have successfully expressed human serotonin receptor 5HT1A in E. coli bacteria, and it was functionally active. Since they have our required range of action, they work by activating adenylyl cyclases, which are present in Ecoli. This can act as a potential sensor. 

( Side effects: 5HT1A can bind to different drugs like antidepressants and LSD)

https://pubmed.ncbi.nlm.nih.gov/1533220/

Histidine Kinases:

Pseudomonas aureginosa possesses a natural serotonin sensor, CPXA, which activates at our required range of serotonin. Serotonin binding with CPXA activates histidine kinases to regulate the expression of underlying genes. So, we wanted to use this sensor to activate our genes for the serotonin-to-melatonin conversion pathway.

(Side effect: CPXA can bind to indole)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351610/

Aptamer sensor:

 There are aptamer sensors that can specifically bind to serotonin. Incorporation of these into Ecoli can be used as a method of activating genetic circuits in our bacteria.

https://doi.org/10.1515/ntrev-2022-0514

We needed to understand how these sensors can be modified to be used for serotonin regulation and which is the most feasible method among these.

We presented our project to Dr. George Church along with the possibilities. He suggested that using the CPXA is the better option as it is easy as it is present directly in the gram-negative bacteria. Furthermore, aptamers require binding domains, which require lots of time to work on, and the 5HT1A receptor mechanism requires more specificity. He mentioned that mutation of cpxA to bind specifically to serotonin is the best option. Another method he suggested is to decrease the sensitivity of Las R using error-prone PCR and make Las R specific to high levels of serotonin.

Integration:

*We used protein docking to modify protein structure to increase specificity. We then docked LasR and Serotonin and found out the residues that bind to Serotonin. We ran a mutational impact analysis on these residues. We thus report various mutants of LasR that have varied sensitivity to serotonin. This allows us to design better and more accurate bio switches in the future that get triggered at various concentrations. Unfortunately, we could not complete the process due to time constraints and the unavailability of resources. 

https://www.linkedin.com/in/aishwarya-nair-99ab8b156/?originalSubdomain=in

We contacted Dr. Aishwarya Nair, co-founder of Mandal Bioworks, a probiotic startup company. Our aim was to evaluate our project from an entrepreneurship perspective; we sought the modifications required to improve our project and transform it into a real product in the market, as well as understand the challenges we might face on the way. We also wanted to understand and analyze the characteristics of the existing probiotics.

Q: What does it take to initiate a start-up for a probiotic product? 

A: “ There are various platforms that support startups, you can pitch your idea and apply for various grants. The most important quality of an entrepreneur is the mindset of consistent effort or perseverance and acceptance of rejections with a positive attitude. Talking to various people in related fields can develop your understanding, and it helps you build your own goals. Once you get into the community, you can figure out rest according to your idea.”

Q: How accepted are probiotics, and what about their marketing demands? 

A: “Probiotics are widely accepted nowadays, there are daily consumers of probiotics from children 4 years to the adult population. Consumer numbers have been increasing over the years at a high rate. In India, people are consuming natural probiotics, though the term familiarity is restricted to a few societies. Indians have a clear understanding of the benefits that probiotics can provide, and very soon, probiotics can take over markets than it is doing right now.”

Q: What are the major challenges that we face in developing a probiotic (related to marketing and entrepreneurship)? 

A: “One of the major challenges is the viability of cells in the gut environment and appropriate storage. For our trials, we tested available probiotics in vitro to see their multiplication, and surprisingly, many of them were not active. In this case, maintaining cell viability until it reaches the large intestine has become a challenging task. Another challenging factor is the competitors in the field. Your product should be better than others and should be chosen by consumers. That is another important factor that comes with developing a product. Human resources involved are difficult to maintain, especially with trustworthy people and in the case of managing funds. Apart from that, proving the efficacy of the product and legal biosafety and ethical clearances involved are challenging.”

Q: What are consumers looking for, and what do you think is lacking in the probiotic industry? 

A: “This is the main point and basis of a successful marketing strategy. This is something to be figured out by yourself as it varies on types of probiotics. Identify your competitors, observe what needs to be added and develop yours. You can engage with stakeholders directly or indirectly to understand this. In our case, price and taste are something that we target. A product that is truly good as claimed and made with an intention to help people will make a difference; that's something lacking in currently existing ones. Consumers are looking for low price, tasty, comfortable consumption methods with quality.”

Q: How far do ethics and morals align with entrepreneurship? 

A: “Speaking very frankly, in most companies, ethics and morals are largely violated for business gains. Still, there are pure minds in emerging entrepreneurs. Further, it depends upon the people, both entrepreneurs and consumers. There should be a sharp mind with brilliant tactics to defy disguised arrows from different directions. In your case, since it is therapeutic, you should have a straightforward approach. However, you cannot avoid certain manipulations that are necessary to move on successfully.”

Q: What marketing channels or strategies have been most effective in promoting probiotics?

A: “A therapeutic needs scientific validation more than anything else. Find doctors and experts related to the field and convince them that your product is good and effective in the treatment of IBS and is better than existing ones. Thereby, you can introduce your product and ask them to prescribe this to patients. Initially, start with medical practitioners and move on to medical shops, etc., to expand your marketing.”

Integration:

Our decision was to develop a drug delivery system that specifically targets the colon, ensuring the survival and effectiveness of the enclosed cells. Additionally, we chose to utilize environmentally friendly and cost-efficient materials for the capsule's outer shell. To enhance consumer comfort during ingestion, we also opted to incorporate a pleasant flavor into the capsule.

https://www.iisertirupati.ac.in/faculty-details/?faculty_id=Che-14&id=2376

We contacted him since solving a set of 9 ODEs with 21+ parameters, was quite tricky and difficult to solve. We needed suggestions for finishing the modelling within the given time.

Integration:

 We figured out that there were too many choke points for the failure of the solution of 9 ODEs. Thus we ended up considering several processes like protein production, serotonin uptake, and induction of promoter as quasi-static. This ended up simplifying the ODEs pleasantly into 3 solvable ones.

https://www.researchgate.net/profile/Arunima-Verma-5

She works with protein-ligand binding. We contacted her seeking help with Proteing docking

Integration.

She introduced us to various software for protein visualisation and docking. Her knowledge of docking and serotonin’s interactions as ligands was extremely helpful in learning about proteins and docking.

https://www.aminer.cn/profile/rajashekar-varma-kadumuri/640500117691d561fb44ab72?source=bz1

We approached him to learn about how to produce mutations in proteins and analyse the results in docking.

Integration:
He introduced us to various ways in which mutagenesis is done computationally. His expertise in docking was using CB-Dock2. We then docked LasR and Serotonin and found out the residues that bind to Serotonin. We ran a mutational impact analysis on these residues. We thus report various mutants of LasR that have varied sensitivity to serotonin. This allows us to design better and more accurate bio switches in the future that get triggered at various concentrations.