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Parts

Introduction

About 1% of the world population is affected by celiac disease8, an autoimmune disorder triggered by the ingestion of gluten, a protein commonly found in wheat, barley, and rye5. This immune response can cause significant intestinal damage from chronic inflammation, nutrient malabsorption, and even lactose intolerance, making it crucial to find effective treatments. This is further underscored by the widespread presence of gluten in the human diet. The UT Austin 2024 iGEM team seeks to alleviate the burden of celiac disease by developing a collection of parts capable of secreting proteases in a bacterium specifically designed to degrade gliadin, the primary immunogenic component of gluten2. By engineering this bacterium to break down gliadin in a sustained and localized manner, the team aims to prevent the harmful effects of accidental gluten ingestion, offering a solution to improve the lives of individuals with celiac disease.

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Our parts collection consists of a diverse array of plasmid backbones (Type 56781), promoters (Type 2), signal peptides (Type 3a), and enzyme coding sequences (Type 3b), designed to enable the modular engineering of plasmids that express gliadin-degrading enzymes. Drawing from the methodologies established in the Yeast Toolkit9 and the Bee Microbiome Toolkit10, our collection allows for the seamless arrangement of genetic parts using type IIS enzymatic Golden Gate Assembly (GGA). Similar to the BTK, our plasmid elements — including broad-host-range promoters, coding sequences, and antibiotic resistance genes — can be independently replaced to optimize performance for specific bacterial hosts.

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Our work includes four areas of focus:

  • Shuttle plasmid backbones in Gram Positive bacteria
  • Weakly constitutive promotors from Antibiotic Resistance genes
  • Gliadin-degrading Enzyme expression
  • Protein Secretion using SecII-dependent Signal tags

The parts in our collection work synergistically to achieve varying levels of constitutive production and efficient protein secretion. To investigate this, we created numerous composite parts to identify optimal promoters and secretion tags, focusing on their transcriptional strength and secretion efficiency. These constructs were then inserted into three domesticated backbones, designed to serve as modular plasmid vectors for ideal functionality.

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Categorization

Basic Parts

Promoters (Type 2)

22 broad-host-range promoters were selected from common antibiotic resistance gene cassettes used in engineered plasmids. Each promoter was tested for its relative strengths with a red fluorescent protein in a pIB184 backbone.

Coding Sequences (Type 3a + 3b)

22 broad-host-range promoters were selected from common antibiotic resistance gene cassettes used in engineered plasmids. Each promoter was tested for its relative strengths with a red fluorescent protein in a pIB184 backbone.

  • Signal tags (3a): Nine Sec-dependent signal tags, previously tested in E. coli or derived from gram-positive bacteria, were paired with fluorescent proteins, and tested for secretion efficiency. They were further evaluated with gliadin-degrading enzymes.
  • Proteins & Proteases (3b): Fluorescent proteins such as mScarlet and sfGFP were used as reporters to assess protein secretion. Well-characterized gliadin-degrading enzymes like Kuma030 and AN-PEP were tested for their activity.

Backbone (Type 56781)

An E. coli expression plasmid and two shuttle vector plasmids with origins that replicate in both E. coli and gram-positive bacteria were modified to create compatible plasmid backbones. They were paired with a green fluorescent protein, signal tags, and gliadin-degrading enzymes.

Composite Parts

Coding Sequences (Type 3a + 3b)

These plasmids were created to assess the efficiency of using different tags to secrete reporter proteins or gliadin-degrading enzymes from bacteria.

Composite Promoter Plasmids

These plasmids were designed to assess the transcriptional strength of the various promoters through fluorescence tests using the iGEM Measurement Kit containing calibration beads for plate readers.

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Submitted Basic and Composite Parts

References

  1. Baldwin, G., Haddock-Angelli, T., Beal, J., Dwijayanti, A., Storch, M., Farny, N., Telmer, C., Vignoni, A., Tennant, R., & Rutten, P. (2019). Calibration Protocol - Plate Reader Fluorescence Calibration v3. ACS Synthetic Biology. https://doi.org/10.17504/protocols.io.6zrhf56
  2. Barone, M.V., Troncone, R., Auricchio, S. Gliadin Peptides as Triggers of the Proliferative and Stress/Innate Immune Response of the Celiac Small Intestinal Mucosa. Int. J. Mol. Sci. 2014, 15, 20518-20537. https://doi.org/10.3390/ijms151120518
  3. Beal, J., Haddock-Angelli, T., Gershater, M., Sanchania, V., Buckley-Taylor, R., Baldwin, G., Farny, N., Tennant, R., & Rutten, P. (2020). Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles v4. iGE. https://dx.doi.org/10.17504/protocols.io.bht7j6rn
  4. Biswas, I., Jha, J. K., & Fromm, N. (2008, August 1). Shuttle expression plasmids for genetic studies in streptococcus mutans. microbiologyresearch.org. https://doi.org/10.1099/mic.0.2008/019265-0
  5. Celiac Disease Foundation. (2024). What Is Celiac Disease? Celiac Disease Foundation; Celiac Disease Foundation. https://celiac.org/about-celiac-disease/what-is-celiac-disease/
  6. Engler C., Kandzia R., Marillonnet S. (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3(11): e3647. https://doi.org/10.1371/journal.pone.0003647
  7. LaFleur, T.L., Hossain, A. & Salis, H.M. Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria. Nat Commun 13, 5159 (2022). https://doi.org/10.1038/s41467-022-32829-5
  8. Lebwohl, B., Sanders, D. S., & Green, P. H. R. (2018). Coeliac disease. Lancet (London, England), 391(10115), 70-81. https://doi.org/10.1016/S0140-6736(17)31796-8
  9. Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology, 4(9), 975-986. https://doi.org/10.1021/sb500366v
  10. Leonard, S. P., Perutka, J., Powell, J. E., Geng, P., Richhart, D. D., Byrom, M., Kar, S., Davies, B. W., Ellington, A. D., Moran, N. A., & Barrick, J. E. (2018). Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids. ACS synthetic biology, 7(5), 1279-1290. https://doi.org/10.1021/acssynbio.7b00399
  11. Murphy, E., Huwyler, L., & de Freire Bastos, M. do C. (1985). Transposon Tn554: complete nucleotide sequence and isolation of transposition-defective and antibiotic-sensitive mutants. The EMBO Journal, 4(12), 3357-3365. https://doi.org/10.1002/j.1460-2075.1985.tb04089.x
  12. Potapov, V., Ong, J. L., Kucera, R. B., Langhorst, B. W., Bilotti, K., Pryor, J. M., Cantor, E. J., Canton, B., Knight, T. F., Evans, T. C., & Lohman, G. J. S. (2018). Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synthetic Biology, 7(11), 2665-2674. https://doi.org/10.1021/acssynbio.8b00333
  13. Sambrook, J., & Russel, D. W. (2001). Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press.
  14. Samperio, S., Guzmán-Herrador, D. L., May-Cuz, R., Martín, M. C., Álvarez, M. A., & Llosa, M. (2021, January 22). Conjugative DNA transfer from E. coli to transformation-resistant lactobacilli. Frontiers. https://doi.org/10.3389/fmicb.2021.606629