Abstract
For the 2023 iGEM competition, the iGEM Guelph team worked towards developing a probiotic to address vitamin B12 deficiency, which is common in older populations, vegetarians, women, and populations from lower and middle-income economies. Supplements or needle injections are the current treatments for this condition, however, older people may not respond as well to supplements because they are unable to absorb the nutrients adequately. Therefore, the BloomBiota project aimed to create a probiotic that contains enriched B12 and human gastric intrinsic factor proteins. Due to the pill nature of BloomBiota, the product could be accessible for a broad range of consumers. This includes vegans, vegetarians, those with gluten intolerances, and those with other intolerances, as the pill casing could be modified based on consumer needs. By taking just one pill, the individual receives a B12 supplement and increased absorption into their cells. We attempted to do this by engineering the B12 cluster and the GIF protein gene fragment into Escherichia coli Nissle 1917 and E. coli DH5ɑ. What made the iGEM Guelph approach novel was attempting to have the bacteria express hGIF in the periplasm, which may aid in proper folding and result in a functional protein. Ultimately, within the timeframe and budget given, the project was unsuccessful. iGEM Guelph was, however, able to contribute several new parts to the registry, and identify future directions for the project. Moving forward, when attempting to transform E. coli Nissle 1917 with a BAC containing the B12 cluster and the plasmid containing hGIF, it would be advisable to use CRISPR to remove the two plasmids pre-existing, allowing facilitated uptake of the large, engineered BAC.
Figure 1: The digestion of BloomBiota and absorption of its products.
Note: The probiotic is taken orally, and travels through the digestive tract with the pill dissolving in the duodenum. From here, E. coli Nissle enters the digestive tract and begins producing both GIF and vitamin B12. Once GIF binds vitamin B12, it can be taken up into the bloodstream at the ileum. This diagram has been adapted from Ferrand et al., 2014, and was created using Biorender.
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Vitamin B12 Deficiency
Figure 2: A visual representation of who vitamin B12 deficiency impacts the most.
Note: This image was made using Biorender
Vitamin B12 is an essential nutrient that is commonly deficient in populations all around the world. Vitamin B12 deficiency contributes to a wide range of clinical disorders. Anemia, neurological, gastrointestinal, and psychiatric disorders are some of the most common related illnesses (Briani et al., 2013). Older populations are at increased risk of vitamin B12 deficiency, and approximately 9.5% of the world population is 65 or older, with the U.N. projecting this number to increase to over 16% by 2050 (Figure 1). The increased prevalence of individuals switching to a vegetarian or vegan diet for health and environmental reasons will also increase the burden of vitamin B12 deficiency. In Canada alone, the number of people who identify as vegetarian or vegan has quickly increased from approximately 900,000 in 2003 to over 3 million in 2018 (Flanagan, 2018). These factors will compound the vitamin B12 deficiency issue across all age groups as the age of vegetarians and vegans skews towards younger members of the population (Flanagan, 2018). Populations in lower-middle-income countries are also at high risk of vitamin B12 deficiency, with a 2019 study from India reporting rates as high as 47% in some cities (Singla et al., 2019).
Figure 3: United Nations projections for the percentage of the world's population that will be 65 years or older from 2023 until 2100.
Figure 4: A visual representation of dementia, mental health disorders, and fatigue, all of which can be linked to vitamin B12 deficiency.
Note: This diagram has been adapted from Ferrand et al., 2014, and was created using Biorender.
Neurological issues caused by a deficiency of vitamin B12 are linked to the breakdown of myelin, which insulates axons to improve neuronal conductivity and messaging (Scalabrino, 2009). This breakdown is caused by increased synthesis of myelinotoxic cytokines, which break down myelin sheath in the central nervous system (Scalabrino, 2009). These physical neurodegenerative effects can lead to documented psychiatric issues such as dementia, mood disorders, psychoses and fatigue (Dommisse, 1991). One of the main mechanisms of dementia-related disorders is the accumulation of homocysteine in the central nervous system (Obeid et al., 2007). This accumulation is caused by a reduction in the function of cystathionine ß-synthase as it requires vitamin B12 as a cofactor to effectively catalyze the breakdown of homocysteine (Obeid et al., 2007).
These are among the more prevalent issues caused by vitamin B12 deficiency, however it is not an exhaustive list of the myriad of biological functions detrimentally affected by this deficiency.
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Human Gastric Intrisic Factor
iGEM Guelph chose to attempt to transform our bacteria with human gastric intrinsic factor (hGIF) because over 60% of elderly individuals with a B12 deficiency have difficulty absorbing vitamin B12 (Andres, 2004). Through the addition of hGIF, our probiotic would be a single product that could not only increase the amount of vitamin B12 in the gut, but also increase its absorption. This way, individuals needing more B12 in their systems wouldn’t necessarily need to figure out the specific cause of their deficiency, but could pick up a single pill as a catch all solution to their deficiency.
Attempting to have E. coli Nissle 1917 express hGIF is part of what makes the BloomBiota project novel, as many bacteria are already able to produce B12 without the intervention of synthetic biology. The human gastric intrinsic factor has already been expressed in the bacterial cytoplasm, however, expression in this location results in the protein needing further refinement to become functional. iGEM Guelph’s goal was to express hGIF in the periplasm of the bacteria, where we hypothesize that it will be able to fold properly, resulting in a functional GIF protein.
Figure 5: Localization of plasmind-expressed GIF in the E. coli Nissle.
Note: In the past, GIF has been expressed in the cytoplasm. BloomBiota aims to express GIF in the periplasm. This diagram was created using Biorender.
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Hardware: The Pill Packing Machine
The Engineering team's primary objective was to address the challenges inherent in outsourcing pill packaging and filling for small startups and research laboratories. These challenges include high costs, extended turnover times, and operational inefficiencies. Our strategy centered on enhancing interchangeability, volumetric control, and overall system adaptability. This approach enables precise customization of volume and dosage, accommodating various characteristics such as powder density and compressibility, and facilitating adjustments for different pill sizes.
iGEM Guelph's innovative solution, the pill packer, features a hybrid capsule orienter-filler-presser system, a rotating wooden circular plate, and a wooden frame. It leverages two pneumatic linear actuator mechanisms: one positioned horizontally for capsule orientation and filling, regulating powder flow, and the other placed vertically for pressing, ensuring proper powder compression. This pill filling machine streamlines operations, guiding the capsule tray through all subsystems until each row contains four fully filled and pressed pills. It represents a user-friendly and efficient approach to pharmaceutical packaging, effectively addressing crucial outsourcing challenges in the industry.
Figure 6: iGEM Guelph's pill packing machine
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More Info
Want to learn more? Check out our Hardware, Experiments, Results, and Future Directions pages!
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References
Andres, E. (2004). Vitamin B12 (cobalamin) deficiency in elderly patients. Canadian Medical Association Journal, 171(3), 251–259. https://doi.org/10.1503/cmaj.1031155
Briani, C., Dalla Torre, C., Citton, V., Manara, R., Pompanin, S., Binotto, G., & Adami, F. (2013). Cobalamin Deficiency: Clinical Picture and Radiological Findings. Nutrients, 5(11), 4521–4539. https://doi.org/10.3390/nu5114521
Dommisse, J. (1991). Subtle vitamin-B12 deficiency and psychiatry: a largely unnoticed but devastating relationship? Medical Hypotheses, 34(2), 131–140. https://doi.org/10.1016/0306-9877(91)90181-w
Ferrand, A., Siu, V. M., Rupar, C. A., Napier, M. P., Al-Dirbashi, O. Y., Chakraborty, P., & Prasad, C. (2015). Biochemical and Hematologic Manifestations of Gastric Intrinsic Factor (GIF) Deficiency: A Treatable Cause of B12 Deficiency in the Old Order Mennonite Population of Southwestern Ontario. JIMD reports, 18, 69–77. https://doi.org/10.1007/8904_2014_351
Flanagan, R. (2018, July 25). More than 3 million Canadians vegetarian or vegan: study. CTVNews; CTV News.
Obeid, R., McCaddon, A., & Herrmann, W. (2007). The role of hyperhomocysteinemia and B-vitamin deficiency in neurological and psychiatric diseases. Clinical Chemical Laboratory Medicine, 45(12). https://doi.org/10.1515/cclm.2007.356
Scalabrino, G. (2009). The multi-faceted basis of vitamin B12 (cobalamin) neurotrophism in adult central nervous system: Lessons learned from its deficiency. Progress in Neurobiology, 88(3), 203–220. https://doi.org/10.1016/j.pneurobio.2009.04.004
Singla, R., Garg, A., Surana, V., Aggarwal, S., Gupta, G., & Singla, S. (2019). Vitamin B12 deficiency is endemic in Indian population: A perspective from North India. Indian Journal of Endocrinology and Metabolism, 23(2), 211. https://doi.org/10.4103/ijem.ijem_122_19
World Population Prospects - Population Division - United Nations. (2022). Population.un.org. https://population.un.org/wpp/Graphs/Probabilistic/PopPerc/65plus/900