Composite

Part:BBa_K5119073

Designed by: Dhruv Aggarwal   Group: iGEM24_Austin-utexas   (2024-10-01)


pBTK1030 + AN-PEP + pelB Secretion plasmid

Through our research, we offer a collection of parts ( BBa_K5119000to BBa_K5119089) that enables researchers to assemble their own plasmid that can replicate in both gram-positive species and E. coli, with the added functionality of secreting enzymes capable of degrading gliadin. Explore the entire collection of parts associated with UT Austin's 2024 iGEM project on the Parts webpage.

Introduction

About 1% of the world population is affected by celiac disease, [3] an autoimmune disorder triggered by the ingestion of gluten, a protein commonly found in wheat, barley, and rye.[4] 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 gluten.[5] 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. For more details, please visit our Project Description.
Figure 1: The UT-Austin 2024 iGEM parts collection. This collection includes twenty-two constitutive antibiotic resistance promoters & RBS (Type 2), nine secretion tags (Type 3a), two reporter proteins and four reporter proteins & enzymes (Type 3b), a rpoC terminator (Type 4), and three plasmid backbones (Type 56781). Created with Biorender.com.

Our parts collection consists of a diverse array of plasmid backbones (Type 56781), promoters & RBS (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 Toolkit[6] and the Bee Microbiome Toolkit,[2] 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. The Ribosome Binding Site (RBS) for all promoters were native to the original antibiotic resistance gene. For all Type 2 parts, the RBS site is included in the individual promoter sequences.
Figure 2: An example of an assembly plasmid containing five part types: a plasmid backbone (Type 56781), a promoter (Type 2), a secretion tag (Type 3a), an enzyme coding region (Type 3b), and a terminator (Type 4). Part Type numbers and overhangs are derived from the Yeast Toolkit[6] and the Bee Microbiome Toolkit[2] and follow their guidelines. Created with Biorender.com.

Our research focuses on four key areas:
  • Shuttle plasmid backbones in gram-positive bacteria
  • Weakly constitutive promoters 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.

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)
    • 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

Composite secretion plasmids – 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.

Part Design and Construction

All composite plasmids have the same rpoC terminator: BBa_K5119034
The pBTK1030 constitutive promoter, PelB secretion tag, and the AN-PEP Enzyme sequence were inserted into the pIB184-GFP backbone. The pIB184-GFP backbone was chosen for its broad-host range compatibility, making it viable in both E. coli and lactic acid bacteria such as Lactococcus lactis. This assembly plasmid was made with the BsaI NEB Golden Gate Assembly Kit.[9] The purpose of this part was to test the efficiency of PelB to secrete the AN-PEP gluten-degrading enzyme from E.Coli DH5a cells, under the control of the pBTK1030 constitutive promoter in our collection.

Characterization

Sequencing of our assembly plasmids showed consistent deletions of 1.2-1.5 kb within the AN-PEP coding sequence. These deletions occurred across all constructs at a similar region, which led us to hypothesize that the issue was due to DNA polymerase slippage, likely caused by repetitive sequences within the AN-PEP gene.

Figure 4: List of Composite Promoter plasmids and Composite secretion plasmids. Basic parts of the same type can be interchanged. The table provides definitions for part symbols in SBOL language. Created with Biorender.com.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 363
    Illegal XbaI site found at 367
    Illegal XbaI site found at 5273
    Illegal XbaI site found at 6321
    Illegal PstI site found at 6700
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 363
    Illegal NheI site found at 981
    Illegal NheI site found at 6491
    Illegal NheI site found at 6514
    Illegal PstI site found at 6700
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 363
    Illegal BglII site found at 378
    Illegal BamHI site found at 344
    Illegal XhoI site found at 374
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 363
    Illegal XbaI site found at 367
    Illegal XbaI site found at 5273
    Illegal XbaI site found at 6321
    Illegal PstI site found at 6700
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 363
    Illegal XbaI site found at 367
    Illegal XbaI site found at 5273
    Illegal XbaI site found at 6321
    Illegal PstI site found at 6700
    Illegal NgoMIV site found at 101
    Illegal AgeI site found at 6776
    Illegal AgeI site found at 6816
  • 1000
    COMPATIBLE WITH RFC[1000]


References

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  3. 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
  4. Celiac Disease Foundation. (2024). What Is Celiac Disease? Celiac Disease Foundation; Celiac Disease Foundation. https://celiac.org/about-celiac-disease/what-is-celiac-disease/
  5. 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
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  12. 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
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  15. Jennings, M. P., & Beacham, I. R. (1990). Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins. Journal of bacteriology, 172(3), 1491–1498. https://doi.org/10.1128/jb.172.3.1491-1498.1990
  16. Ahmadi, Z., Farajnia, S., Farajzadeh, D., Pouladi, N., Pourvatan, N., Karbalaeimahdi, M., Shayegh, F., & Arya, M. (2023). Optimized Signal Peptide for Secretory Expression of Human Recombinant Somatropin in E. coli. Advanced pharmaceutical bulletin, 13(2), 339–349. https://doi.org/10.34172/apb.2023.037
  17. Benson, S. A., & Silhavy, T. J. (1983). Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12. Cell, 32(4), 1325–1335. https://doi.org/10.1016/0092-8674(83)90313-6
  18. Bowers, C. W., Lau, F., & Silhavy, T. J. (2003). Secretion of LamB-LacZ by the signal recognition particle pathway of Escherichia coli. Journal of bacteriology, 185(19), 5697–5705. https://doi.org/10.1128/JB.185.19.5697-5705.2003
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