HumanPractices

Gluten Free Community Outreach

Zucchini Kill

One of our first meetings was with Cece, the owner of Zucchini Kill, which is a dedicated gluten-free and vegan bakery located in Austin. During this meeting, we were introduced to the struggles of celiac patients. Cece stressed the idea that people with celiac disease often live in fear because even the smallest amount of gluten can have drastic effects on their bodies for several days. Cece herself lives with gluten intolerance and emphasized how fulfilling it is to be able to provide dessert options to people with celiac disease, especially to little kids with celiac who have never been able to experience dessert before. During this meeting, we also learned that although it seems like Austin has a lot of gluten-free options, it has been getting more difficult for gluten-free businesses to stay open, given the high cost of gluten-free ingredients and their relative unavailability. Cece highlighted that in order to stay open, gluten-free businesses must provide products that have comparable quality and taste to normal gluten products. Cece even gave us some insight on her unique blend of ingredients she uses to make gluten-free flour, including sorghum, tapioca, chickpea, white and brown rice, xanthan gum, and almonds. Cece, like many other bakers we would learn about, often has to acquire these novel ingredients from specialty suppliers, which is neither affordable nor easy to procure. Although there has been increased awareness of food allergies and more people have been discovering that they feel better when they eat gluten-free foods, the options for this community are declining, which makes our project even more important.

Bread(ish)

Continuing our interactions with the gluten-free community, we met with Lisa Shannon, the owner of the gluten-free pop-up bakery Bread(ish). Lisa gave us another crucial piece to the story of our project. Lisa explained that there is a general lack of options for people with a gluten-free diet, and with the limited options available, cross contamination still plays a factor in deciding what to eat, especially when eating out. Lisa has made it her goal to provide the Austin gluten-free community with gluten-free artisanal breads because as a person whose entire family is gluten-free, it was important to her. Lisa also mentioned to us that although she appreciates us working on solutions, she believes that people with celiac disease would be too afraid of the consequences of consuming gluten again, even with our product. With this in mind, we started considering that it might be better to focus our product on eliminating the effects of cross contamination rather than allowing people with celiac disease to include gluten in their diet again. Although we hope to eventually increase our probiotic efficiency, this meeting helped us realize that guarding against cross contamination would be a more feasible goal.

Figure 4. Meeting with owner of Bread(ish) bakery.

Living with Celiac 1

Stayce King's journey with celiac disease has been a long and challenging road marked by resilience and adaptation. Diagnosed only recently in February of 2024, Stayce had likely been living with the condition for over 20 years. Her path to diagnosis was complicated by her Multiple Sclerosis, which initially masked her digestive symptoms, making it difficult to pinpoint the exact cause of her discomfort. This long period of uncertainty is a common story among celiac patients, highlighting the often delayed diagnosis due to the complexity of autoimmune diseases.

Living with celiac disease has significantly impacted Stayce's daily life. She faces the constant challenge of avoiding gluten, which is no easy feat given the pervasive nature of gluten in food and even some non-food products such as hair sprays. Dining out is particularly stressful, with only a handful of places she considers as semi-safe, yet even these carry the risk of cross-contamination. The cost of maintaining a gluten-free diet is another burden, as gluten-free foods are significantly more expensive than their regular counterparts. Additionally, the necessity to replace small appliances to avoid gluten contamination at home adds to the financial strain.

Upon contamination, Stayce's symptoms are consistent and debilitating, including migraine headaches, itching, and severe gastrointestinal issues that can persist for days. Despite these challenges, she has shown remarkable resilience. Stayce leverages her past experience as a baker by opening a gluten-free bakery, turning a personal challenge into a business that helps others with similar dietary restrictions.

She is also open to advancements in treatment and prevention of cross-contamination effects. Stayce expressed interest in a proposed probiotic that could degrade gluten, although she questioned whether it would prevent intestinal damage or merely alleviate symptoms. She is aware of the potential concerns some might have about genetic engineering, but she is personally unbothered by it. Her experience with existing gluten-degrading supplements like Glutenease has been moderately positive, although she finds its effectiveness limited.

Stayce's story is one of perseverance and adaptation in the face of ongoing health challenges. Her journey emphasizes the importance of awareness, accurate diagnosis, and innovative treatments in improving the lives of those with celiac disease. As she continues to navigate through her condition, Stayce remains a hopeful and active participant in finding better solutions for herself and the broader celiac community.

Figure 5. Meeting with owner of Bread(ish) bakery.

Living with Celiac 2

At 24 years old, Hailey is already well-acquainted with the daily struggles that come with living with celiac disease. Diagnosed in February after years of feeling unwell, Hailey's journey has been anything but typical. Unlike many others with celiac disease, she doesn't experience the clear relief that others often feel after going gluten-free. In fact, her symptoms have remained frustratingly persistent, leaving her to question what “normal” is supposed to feel like.

One of the first challenges Hailey faced after her diagnosis was adjusting to a gluten-free diet. While it promised to be the key factor to feeling better, the reality of finding gluten-free foods that she liked and could afford was daunting. “The bread is like half the size of normal bread, and it's so expensive,” she explains. On top of that, gluten-free food often doesn't last as long. In the beginning, she found herself wasting copious amounts of food as things would get moldy after just a couple of days.

For Hailey, grocery shopping and meal planning are tasks she dreads. Not only are the options limited, but the high cost of gluten-free products adds financial stress to her already challenging condition. The fact that she lives alone complicates things further. Without someone else to compare notes with, she's never entirely sure if she's experiencing cross-contamination. Despite her best efforts, she feels no different after eating gluten-free. Hailey often describes herself as “not the everyday celiac.” Her symptoms didn't align with the typical presentation, making her journey to diagnosis a long and frustrating one. Even after months on a gluten-free diet, she doesn't feel any better. This disconnect from the expected narrative of relief leaves her feeling isolated and uncertain about her path forward.

Adding to her challenges is Ehlers-Danlos syndrome (EDS), a connective tissue disorder that further complicates her life. EDS has brought on a “passing out” disorder, which has only worsened since she went gluten-free. Simple tasks like cooking become monumental challenges because she can't physically stand for long periods or manage the repetitive movements required in the kitchen.

Despite these struggles, Hailey is determined to find a way to manage her conditions and live a fulfilling life. She's aware that for others with both EDS and celiac disease, it took two to three years to start feeling better. This knowledge gives her hope that improvement may still be on the horizon, even if the journey might take longer than she had expected.

Hailey is still new to the celiac community and is navigating the landscape of gluten-free options. She's found a few reliable places, like Jersey Mike's, which offers gluten-free bread, but she's still exploring what is available. “I don't know many other options just because I'm new to the community,” she admits. When asked about the possibility of new treatments, such as a probiotic designed to prevent the effects of cross-contamination, Hailey is optimistic. She believes that the celiac community is generally well-educated and open to innovations, including those involving genetic engineering.

Despite her enthusiasm for new possibilities, Hailey hasn't tried any gluten-degrading pills or drinks. “I was told they don't actually work,” she shares, explaining her reluctance to invest in something that might not deliver on its promises. Hailey's journey with celiac disease is still unfolding. She's learning more about her body, her condition, and the tools available to help her live a healthier, more comfortable life. While the road has been rocky and full of unexpected twists, she remains hopeful that with time, knowledge, and the support of a community, she'll find her way to better days.

Figure 6. Meeting with Hailey Laskoswski, discussing her journey with celiac.

Living with Celiac 3

Holly's life with celiac disease is a constant balancing act, marked by daily challenges that have shaped her lifestyle since she first recognized symptoms in 2006. Holly's path to understanding her condition was not straightforward. She was diagnosed with celiac disease in 2006 after years of suffering from constipation, vomiting, and other systemic symptoms. At the time, she didn't have health insurance, so she took matters into her own hands and went gluten-free on her own, which led to significant improvements. However, because she refused to reintroduce gluten for the sake of a formal diagnosis, she was never officially diagnosed until she underwent a genetic test in 2011. The test confirmed she had the HLA DQ antigens associated with celiac Disease, but by then, her symptoms and response to a gluten-free diet had already convinced her and her GI doctor that she had celiac.

Holly has come to terms with her condition and has found ways to manage it, though it's not always easy. She also avoids dairy, adding another layer of complexity to her diet. She's learned to navigate the world of gluten-free eating, finding enough options in stores to feel satisfied with her choices.

When Holly leaves the house, the reality of living with celiac disease hits the hardest. Simple activities, like going to church, become stressful because she can't participate in snack time. The food provided is often not labeled properly, leaving her to wonder whether it's safe to eat. This isolation during communal events is a constant reminder of her condition. Eating out is another challenge. Despite the growing availability of gluten-free (GF) options, Holly rarely dines out due to the risk of cross-contamination. She experiences it about once or twice a year, and when it happens, her symptoms vary depending on how much gluten she ingests. A small amount might give her a migraine or upset stomach, but significant exposure can lead to hours of vomiting.

Cooking for herself and her kids, who also have celiac disease, has become a routine part of Holly's life. She went gluten-free in 2006, at a time when GF options were scarce, making meal preparation a challenge. However, living in Austin, near Kyle, has its perks. She now finds a good variety of GF products at stores like Walmart and HEB, and she's noticed that GF options have become more mainstream over the years. Despite the improved availability, cooking still requires careful attention to detail. Holly has to ensure that every ingredient is gluten-free, and she constantly worries about cross-contamination in her kitchen. She's learned to read labels meticulously and has become a master at adapting recipes.

When it comes to potential new treatments like a probiotic designed to prevent the effects of cross-contamination by secreting a gluten-degrading enzyme, Holly is cautiously optimistic. She recognizes that there's an audience for such a product, especially for those who are younger, like her child, who might be willing to try a pill if it makes them feel better. However, Holly herself is more focused on addressing the underlying problem through diet rather than relying on a pill. Shes open to the possibilities of gene therapy for celiac but prefers to manage her condition through strict adherence to a gluten-free diet.

Her journey is a testament to her resilience and determination to live a full life, even in the face of a chronic condition. Through it all, Holly remains hopeful and grounded, always striving to find the best path forward for herself and her loved ones.

Figure 7. Meeting with Holly Urbach, discussing her journey with celiac.

Academic Outreach

Dr. Kristina Tzartzeva

Early on in our project we broadly defined our project goal as preventing a gluten allergy immune response. We quickly learned through a meeting with Dr. Kristina Tzartzeva that gluten allergies were not as clear cut as we originally thought. Dr.Tzartzeva is a gastroenterologist at the Ascension Seton Medical Center in Austin, and our meeting with her helped us to define that gluten degradation could make the most impact. She explained the differences between gluten intolerance/sensitivity and celiac disease, the alleged “gluten allergy” we were searching for.

Non-celiac gluten sensitivities/intolerances commonly lead to feelings of sickness after ingesting gluten, including nausea, headaches, and indigestion among other non-immunogenic linked symptoms. While this condition is more common than celiac (affecting about 6% of the US pop.), its effects are less severe and more manageable than celiac disease.

Celiac disease is the true focus of our research project this year, and it affects about 1% of the world's population, causing inflammation and degradation of the intestinal lining due to the ingestion of gluten in afflicted individuals. The disease occurs in those with a genetic haplotype characterized as HLA-DQ2 or HLA-DQ8, which leads to the antibody-mediated response commonly known to cause inflammation. Although celiac disease only occurs in a small fraction of the world's population, it is often misdiagnosed or undiagnosed in up to 83% of cases. Additionally, one study found the rate of new diagnosis of celiac disease has increased 7.5 percent each year for the past few decades. This means that the prevalence of celiac disease is actually much higher than it seems, which makes it increasingly important to bring awareness to this disease and its symptoms.

Figure 8. Rates of diagnosed vs misdiagnosed or undiagnosed celiac disease patients.

In our discussions with Dr.Tzartzeva, she highlighted how patients diagnosed with celiac disease are severely restricted in their dietary options. Avoiding gluten and gluten contamination while finding food alternatives is often a daily struggle for many, as we learned in our earlier meeting with the owner of gluten free bakery Zucchini Kill. Gluten is a large part of the Western diet, with the average American consuming 10-20 grams of gluten per day. Gluten-free options are also often expensive and hard to find. To date, the only approved treatment for celiac disease is a gluten-free diet, with no effective medications currently on the market.

Figure 9. Choosing gluten as the target, as a grain that is notoriously hard to avoid.

After educating us on the problem, Dr.Tzartzeva gave us a push in the right direction toward creating a foundation for our project's main product. She endorsed the idea of using a probiotic to formulate an enzyme therapy that could degrade gluten before it reaches the small intestine, the site of the immune response. A probiotic, as she highlighted, would provide a more convenient, long-term therapy for celiac patients, aiming to relieve some of their daily concerns with managing the disease.

Arbitrarily degrading gluten wouldn't solve the issue though; we needed to know what to degrade, and how to do it efficiently. This was crucial not only for optimizing our project, but also because celiacs would only trust a probiotic that could lay their fears to rest. Celiac patients wouldn't want to take the risk of a product that worked any less than 99% of the time, a point that was similarly highlighted by Stayce in our meeting with her earlier. Dr. Tzartzeva proclaimed that patients suffering from gluten sensitivities and intolerances would possibly prefer enzyme therapy as a new alternative compared to food processing or probiotics. This is the case because many patients are inconsistent with taking the supplement and frustrated with the idea of ingesting new bacteria to acclimate in the gut microbiome. We of course took her insight into consideration as we sought to overcome these challenges and find a way to reach our target audience.

Dr. Ingrid Pultz

In our research on gluten, we discovered the major subcomponent gliadin, a gluten-peptide part of a class of proteins called prolamins which is responsible for the majority of the immunogenic responses in people with celiac disease and gluten intolerances. As we further explored gliadin and several of its constituent immunogenic epitopes, we found structural similarities with proteins found in other grains such as avenin from oats, secalin from rye, and hordein from barley. These analogous protein sequences piqued our interests as potential other targets to include within the scope of our project design. We soon reached out to Dr. Ingrid Pultz, a translational advisor at the Institute of Protein Design and advisor of the University of Washington 2011 iGEM team, to look into the potentials of gliadin targeting enzymes. This team worked on developing Kumamolisin, an extremely efficient gliadin specific gluten-degrading enzyme that is currently in stage 2 clinical trials under Takeda Pharmaceuticals. We learned from our meeting with Dr. Pultz that enzyme specificity is extremely important for efficient gluten degradation. This brought us back to thinking about our meeting with Stayce King, when she told us that the effectiveness of the gluten degrading enzyme pills was limited. After researching the gluten degrading pill she mentioned, we found that the enzymes in it were nonspecific for gliadin, which could be why they were not as effective. Moving forward with our project, we will make sure to integrate an enzyme that is gliadin specific, since that is the subunit of gluten that triggers a reaction in people with celiac disease. In our meeting with Dr. Pultz, we also asked about modifying Kuma to degrade avenin, secalin, or hordein. However, Dr. Pultz emphasized that it is only worth it to degrade proteins that have a disease associated with it. Since the number of people who suffer from allergies or other problems with avenin, secalin, or hordein is small, we decided to keep our focus on degrading gliadin, the primary trigger for celiac disease. However, in the future, we hope that our modular project design can be modified to also target other immunogenic proteins with structures similar to gliadin.

In addition to helping us finalize our target for our project, Dr. Pultz encouraged our idea of using a probiotic, explaining how a probiotic would fill in an avenue for potential treatments not currently being undertaken with Kuma. She also highlighted that the site of degradation would be a crucial element for the success of our proposed idea because if we were to secrete our modified enzyme as late as the small intestine, it would likely be too late. Not only would the gut be too late for most degradation to take place, but finding a bacteria to colonize this region would also pose a challenge. After this meeting, we began considering our options for which host organisms we would use and where they would secrete their gluten degrading enzymes.

Tricia Thompson and Dr. Katherina Scherf

While exploring which gluten prolamin degradation method would be most effective, as well as potential enzyme delivery pathways, we consulted Tricia Thompson, founder of Gluten Free Watchdogs and an advocate for individuals with gluten intolerances and celiac disease. Her initiative focuses on providing state-of-the-art gluten-free food testing data directly to consumers. She referred us to Dr. Katherina Scherf, who leads the Department of Bioactive and Functional Food Chemistry at the Institute of Applied Biosciences, Karlsruhe Institute of Technology (KIT) in Karlsruhe, Germany. Dr. Scherf highlighted that food processing might not be the best therapeutic method to degrade gluten due to changes in taste and the baking properties of gluten-containing products. She discussed the pros and cons of food processing compared to peptidase pills, which helped clarify the direction of our project. She also emphasized the availability of many characterized glutenases from the oral and gut microbiome and suggested utilizing naturally gluten-degrading bacteria that use gluten as an energy source. This insight led us towards incorporating lactic acid bacteria and Rothia, an oral resident bacteria, into our project, and shifted us away from food processing as a delivery site for our gluten-degrading bacteria.

Figure 10. Meeting with Dr. Katherina Scherf.

Project Design and Novelty

Cultivarium

From here, we had narrowed our project design to creating a gut or mouth-resident bacteria that could secrete our gluten-degrading enzyme within a probiotic.Rothia spp. in particular had captivated our interest as it held strong potential for its novelty, inherent gluten degrading capabilities, and natural colonization of the oral microbiome.Rothia species have remained largely unexplored in the field of synthetic biology, and we felt our work could begin to tap into this frontier of research. We weren't the only ones looking to explore the potential of Rothia spp. either, as the species first came to our attention when we found Cultivarium, a non-profit bio-research company looking to sponsor iGEM teams working with this non-model organism. Our iGEM team was fortunate enough to win their sponsorship, and in doing so we had the opportunity to meet with some of Cultivarium's research team who gave us some valuable advice on working with these bacteria.

In our meeting with Cultivarium experts, they advised our transformation and secretion subgroup teams primarily on how to conjugate our modified plasmids into gram positive bacteria, including Rothia mucilaginosa, which is the strain that was offered by their sponsorship. We had previously been having trouble both electroporating and conjugating our plasmids into Rothia mucilaginosa, as nothing would grow on the selective plates. Cultivarium experts let us know that they had not successfully been able to conjugate or electroporate DNA into Rothia strains either. However, some options for troubleshooting included checking the compatibility of our oriT sequence, checking for restriction enzyme sites in the strain that might target our plasmid, and checking for expression of the resistance marker. However, despite trying out methods Cultivarium recommended for conjugating plasmids into Rothia spp., both conjugation and electroporation continued to fail in Rothia mucilaginosa. This led to our decision to ultimately abandon Rothia spp. as a bacterial chassis for our probiotic. We hope to revisit working with the organism in the future as we explore further methods for transforming into this novel synthetic biology strain.

Dr. Neel Joshi

Around this same period of time, our secretion team also met with an expert on secretion systems Dr. Neel Joshi, who had experience testing the Sec/SRP system in E. coli with sfGFP as a reporter gene. His experimental design was nearly identical to our own, so we were excited to converse about the successes and challenges of our work thus far and plans with our project goal in mind. During our meeting, we asked him several questions relating to how we could optimize the secretion efficiency of our current system. Dr. Joshi highlighted the limitations of our current study and gave us several points to consider as we moved our secretion machinery from E. coli to the gram positives strains L. lactis and L. plantarum. Up until this point we had been testing the fluorescent proteins sfGFP and mScarlet in parallel with E. coli native signal peptides and measuring their supernatant and cellular fluorescence fractions as an indicator of the SPs secretion efficiency. However, Dr. Joshi confirmed our suspicions regarding our sfGFP constructs, sfGFP had been exhibiting minimal to zero fluorescence readings. SfGFP folds exceptionally quickly in compared to mScarlet, which we hypothesized was posing a problem in sfGFP secretion since Sec/SRP signal peptides are specialized for secreting unfolded protein products. With this in mind, our secretion team changed its focus towards measuring secretion fluorescence using mScarlet. Dr. Joshi also emphasized that our SP collection would not function with the same efficiency in L. lactis and L. plantarum, a fact we were aware of through our search through literature. However our primary goal through testing with fluorescents proteins was to establish a system to quantitatively measure and confirm the utility of our SP sequences with recombinant proteins. Nevertheless, the secretion team took this meeting as the initiative to begin further integrating our secretion system with our enzyme and transformation teams constructs to move towards our ultimate goal of secreting glutenases within gram-positives.

Dr. Dolores Lucía Guzmàn Herrador

In addition to our secretion team's meeting, our transformation team was fortunate enough to meet with two different experts on transforming lactic acid bacteria in order to troubleshoot transformation into our other main strains to be used as chassis: Lactiplantibacillus plantarum, Lactococcus lactis, and Lacticaseibacillus casei. First, we met with Dr. Dolores Lucía Guzmàn Herrador, who is a postdoc that has worked with conjugating plasmids into Lactobacilli. Dr. Herrador is the author of one of the papers that our transformation team used the protocols of, titled “Conjugative DNA Transfer from E. coli to Transformation Resistant Lactobacilli.” Before our meeting with Dr. Herrador, we had only had successful conjugation of our plasmids from E. coli to E. coli. Our meeting with Dr. Herrador focused on a detailed discussion of the protocol and any discrepancies with the oriT sequence we had cloned into our broad host range plasmids. Dr. Herrador gave us insight that we needed to decrease the concentration of antibiotic on our plates after conjugation and encouraged us to try a new oriT sequence that would work better for conjugation into gram positive bacteria.

Figure 11. Meeting with Dr. Dolores Lucía Guzmàn.

Dr. François Bringel

Second, we met with Dr. Bringel, who is a research director for the French national research. She has previously focused on electroporating DNA into Lactobacillus plantarum, and she wrote the protocol that we were following to electroporate our plasmids into gram positive bacteria. During our meeting with Dr. Bringel, we asked her for tips on why our electroporation trials were arcing, which is when electricity sparks from too many salts present, and kills the cells. We also asked for expertise on why many of the strains that we electroporated with selective plasmids were only growing on plates with no antibiotic. We had previous suspicions that the reason for our electroporation trials arcing had to do with the wash steps of preparing our electrocompetent Lactobacillus plantarum, as the pellet of cells often started dissolving before we could remove the wash solution. When we brought this up to Dr. Bringel, she emphasized that her electroporation success was very strain dependent. She told us that even though we were using the same species, our specific strain of Lactobacillus plantarum might not be suitable. She offered to send us a sample of her specific Lactobacillus plantarum strain, and we have been working to utilize this strain in our electroporation trials.

Dr. Jay Yepuri

As we finalized the framework for our modular system, we felt it was imperative to secure at least one more expert who could corroborate our proposed design method into practice. We were fortunate enough to meet with Dr. Yepuri, an expert on probiotics. During our meeting with Dr. Yepuri, we asked him about what qualities are factored into using specific species of bacteria as probiotics, and the benefits of taking probiotics on gut health. Dr. Yepuri corrected some of our misconceptions by emphasizing that there is no real study that correlates taking probiotics to therapeutic effects on human health. However, he stated that since the main focus of our project is on secreting a gluten degrading enzyme from this bacteria, the specific gut health benefits of the bacteria chosen should not matter. Dr. Yepuri recommended using a bacterial strain that is already used commercially and deemed safe by the FDA, which our strains of lactic acid bacteria all fit the criteria for. Dr. Yepuri also mentioned that all gut microbiomes are different, and although our probiotic may colonize the gut of some people, it may have to be taken more regularly by the majority of the population. Testing will have to be conducted to determine the best time scale for best efficacy of our gluten degrading probiotic. Furthermore, Dr. Yepuri believes that the introduction of an engineered probiotic would be widely accepted in America, and he recommended developing our probiotic to be delivered orally through a pill. This would allow the bacteria to survive past the extremely acidic pH of the stomach and allow for better colonization. As we continue to develop our probiotic, we will take Dr. Yepuri's advice into account.

Figure 12. Meeting with Dr.Yepuri (GI Doctor).

Human Practices Major Takeaways

Overall, the work of our human practices team was key for gaining feedback on both our wet lab methods and the needs of the celiac disease community, which is a major project stakeholder. As we continued to engage with the celiac community and scientific experts, we continually adjusted our proposed solution to the needs of the community and the practicality of the wet lab work. From these conversations, we left with a few major takeaways:

1. A gluten-free diet is the only effective treatment for celiac disease, but it is expensive and hard to maintain. Even though the number of people going gluten-free is increasing, gluten-free bakeries and businesses in Austin are declining due to expensive ingredient costs.
2. Celiac disease is often debilitating, and even trace amounts of gluten can cause extreme symptoms. Therefore, it is unlikely that people with celiac disease will be able to include gluten in their diet again. However, our probiotic will be able to serve as a guard against cross contamination, which is a common problem that people with celiac disease have to live with when going out to eat.
3. Gluten has to be degraded before reaching the small intestine, which is the site of inflammation in people with celiac disease. Our bacteria strains: L. lactis, L. plantarum, L. casei, and Rothia mucilaginosa are all gastrointestinal tract bacteria, which should secrete a gluten degrading enzyme before reaching the small intestine.