Coding

Part:BBa_K5119027

Designed by: Dhruv Aggarwal   Group: iGEM24_Austin-utexas   (2024-09-27)


PhoE Secretion Tag

This part is a native signal peptide in the Sec/SRP pathway originally derived from the PhoE gene in E. coli.[23] 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 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.

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.

Usage and Biology

This part is a 63 bp sequence that encodes for a secretion peptide that is attached to translated proteins. Signal peptides guide these proteins to extracellular secretion in different ways, and this tag uses the Sec/SRP pathway to achieve secretion. The PhoE secretion tag peptide was previously investigated to determine its usefulness in the secretion of recombinant proteins [16]. The team acquired the PhoE sequence from IDT. One subproject of the UT Austin iGEM 2024 team aimed to assess the secretion efficiency of common signal tags in specific backbones used in engineered plasmids. This involved constructing a library of signal peptides, integrating them into modular plasmid systems, and using fluorescent proteins like sfGFP and mScarlet to quantify their secretion efficiency. We systematically tested these signal tags in E. coli using the domesticated pET28a and pIB184 backbones. Overall, the team sought to identify optimal signal peptides that could facilitate the secretion of gliadin-degrading enzymes, contributing to the development of a broad-host range secretion system.

Associated Composite Parts



Figure 3: 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.

Part Design and Construction

To ensure optimal performance in E. coli, the PhoE codon sequence was optimized, and the prefix and suffix were designed as Type 3a parts following the standards of the Yeast Toolkit and Bee Microbiome Toolkit. The PhoE secretion signal tag was then integrated into the pET28a backbone to create the composite secretion plasmids parts that also contain a fluorescent protein sequence (mScarlet or sfGFP) and a rpoC terminator. This assembly plasmid was made with the BsaI NEB Golden Gate Assembly Kit (GGA) [9]. These composite plasmids were designed to facilitate the extracellular secretion of fluorescent proteins in E. coli Tuner DE3. The GGA product was sequenced and confirmed through Plasmidsaurus Whole Plasmid Sequencing.
We used a microplate reader to measure the fluorescence in the supernatant, which allowed for the quantification of secretion efficiency. The ultimate aim was to use PhoE to enable the secretion of gluten-degrading enzymes.

Characterization

The initial results for PhoE-tagged constructs indicated that their secretion efficiency was variable. In the pET28a plasmid, one of the challenges encountered was the high transcription rate of the T7 promoter, which led to instability and inconsistent secretion. Although fluorescence was observed in the supernatant, suggesting some level of secretion, the efficiency was not significantly higher than the controls. The instability of the pET28a plasmid due to the T7 promoter’s high transcription rate appeared to affect the performance of the PhoE signal peptide. Additionally, there were signs of cell lysis in some of our assays, which could have contributed to the observed fluorescence in the supernatant, complicating the interpretation of the data. Overall, the findings suggested that some signal peptides, including PhoE, may need further optimization to improve secretion in different bacterial strains, particularly when transitioning to gram-positive bacteria like Lactococcus lactis.

Figure 4. Secretion of mScarlet when paired with secretion tags in pET28a. Secretion efficiencies and amount of fluorescence generated in the extracellular milieu relative to the number of cells across several trials of induction. A) Supernatant fluorescence over culture fluorescence B) Supernatant fluorescence over culture fluorescence. Data for each signal tags averaged out across 2 trials for each induction group. Fluorescence measured at one timepoint from a micro plate reader using cultures grown for 20 hours of incubation at 37 °C following our induction protocol. Supernatant fluorescence taken from supernatant of cells spun down at 7000 rpm at 4°C for 10 mins.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. Novick, R. P., & Murphy, E. (1985). MLS-resistance determinants in Staphylococcus aureus and their molecular evolution. Journal of Antimicrobial Chemotherapy, 16(suppl A), 101–110. https://doi.org/10.1093/jac/16.suppl_a.101
  2. 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
  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
  6. 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
  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. 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
  9. 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
  10. Sambrook, J., & Russel, D. W. (2001). Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press.
  11. 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
  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
  13. Biswas, I., Jha, J. K., & Fromm, N. (2008). Shuttle expression plasmids for genetic studies in Streptococcus mutans. Microbiology (Reading, England), 154(Pt 8), 2275–2282. https://doi.org/10.1099/mic.0.2008/019265-0
  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
  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
  19. Takahara, M., Hibler, D. W., Barr, P. J., Gerlt, J. A., & Inouye, M. (1985). The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli. Journal of Biological Chemistry, 260(5), 2670–2674. https://doi.org/10.1016/S0021-9258(18)89413-3
  20. Li, Z., Leung, W., Yon, A., Nguyen, J., Perez, V. C., Vu, J., Giang, W., Luong, L. T., Phan, T., Salazar, K. A., Gomez, S. R., Au, C., Xiang, F., Thomas, D. W., Franz, A. H., Lin-Cereghino, J., & Lin-Cereghino, G. P. (2010). Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris. Protein expression and purification, 72(1), 113–124. https://doi.org/10.1016/j.pep.2010.03.004
  21. Pechsrichuang, P., Songsiriritthigul, C., Haltrich, D., Roytrakul, S., Namvijtr, P., Bonaparte, N., & Yamabhai, M. (2016). OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. SpringerPlus, 5(1), 1200. https://doi.org/10.1186/s40064-016-2893-y
  22. Power, B. E., Ivancic, N., Harley, V. R., Webster, R. G., Kortt, A. A., Irving, R. A., & Hudson, P. J. (1992). High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PelB secretion signal. Gene, 113(1), 95–99. https://doi.org/10.1016/0378-1119(92)90674-E
  23. de Cock, H., Overeem, W., Tommassen, J. (1992). Biogenesis of outer membrane protein PhoE of Escherichia coli: Evidence for multiple SecB-binding sites in the mature portion of the PhoE protein. Journal of Molecular Biology, 224(2), 369-379. https://www.sciencedirect.com/science/article/pii/0022283692910016?pes=vor
  24. Adams, H., Scotti, P. A., De Cock, H., Luirink, J., & Tommassen, J. (2002). The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal-recognition particle in Escherichia coli. European journal of biochemistry, 269(22), 5564–5571. https://doi.org/10.1046/j.1432-1033.2002.03262.x
  25. Ravn, P., Arnau, J., Madsen, S. M., Vrang, A., & Israelsen, H. (2000). The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis. Gene, 242(1-2), 347–356. https://doi.org/10.1016/s0378-1119(99)00530-2
  26. Ravn, P., Arnau, J., Madsen, S. M., Vrang, A., & Israelsen, H. (2003). Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis. Microbiology (Reading, England), 149(Pt 8), 2193–2201. https://doi.org/10.1099/mic.0.26299-0
  27. Morello, E., Bermúdez-Humarán, L. G., Llull, D., Solé, V., Miraglio, N., Langella, P., & Poquet, I. (2008). Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion. Journal of molecular microbiology and biotechnology, 14(1-3), 48–58. https://doi.org/10.1159/000106082
  28. Ng, D. T., & Sarkar, C. A. (2013). Engineering signal peptides for enhanced protein secretion from Lactococcus lactis. Applied and environmental microbiology, 79(1), 347–356. https://doi.org/10.1128/AEM.02667-12


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