Part:BBa_K5119017

SpcR FL Promoter

This part is a full-length spectinomycin antibiotic resistance promoter originally found in Staphylococcus aureus [1] and obtained from pBTK403, a broad-host-range bacterial origin plasmid (Leonard et al., 2018). Through our research, we offer a collection of parts ( BBa_K5119000to BBa_K5119087) 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 (Lebwohl et al., 2018), an autoimmune disorder triggered by the ingestion of gluten, a protein commonly found in wheat, barley, and rye (Celiac Disease Foundation, 2024). 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 (Barone et al., 2014). 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 22 constitutive antibiotic resistance promoters (Type 2),9 secretion tags (Type 3a), 2 reporter proteins and 4 reporter proteins & enzymes (Type 3b), a rpoC terminator (Type 4), and 3 plasmid backbones (Type 56781). Created with Biorender.com.

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 Toolkit (Lee et al., 2015) and the Bee Microbiome Toolkit (Leonard et al., 2018), 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. 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). The table provides definitions for part symbols in SBOL language. 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.
• Protein secretion using SecII-dependent signal tags

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

SpcR is a specific antibiotic resistance gene that provides resistance to spectinomycin antibiotics. For this gene to operate at a high level, it is upregulated by the SpcR promoter.

This SpcR promoter and SpcR gene are found in pBTK403, a plasmid from the Bee Tool Kit. The pBTK403 plasmid was originally designed and used to engineer the bee gut microbiome bacteria to improve bee health (Leonard et al., 2018). A combination of genetic parts was used for the modular construction of broad-host-range plasmids, including pBTK403, using the RSF1010 replicon. The pBTK403 plasmid was obtained from the Barrick Lab.

One subproject of the UT Austin iGEM 2024 team aimed to assess the transcriptional strengths of various promoter regions found in common antibiotic resistance cassettes used in engineered plasmids. These promoters are broad-host-range, meaning they are compatible with multiple bacteria species. Hence, characterizing their relative strengths can prove useful, as these promoters can be incorporated into the plasmid designs of other subprojects of the UT Austin iGEM 2024 team.

Associated Composite Parts

Figure 3: List of Composite Promoter plasmids and Composite secretion plasmids. Basic parts of the same type can be interchanged. Created with Biorender.com.

• SpcR FL + mScarlet Promoter plasmid – BBa_K5119050

Experimental Design

De Novo DNA's promoter calculator was used to predict nucleotide positions in the SpcR promoter region with high transcription rates (LaFleur et al., 2022). High transcription rates may indicate potential promoter start sites. These predicted sites were used to design five variants of the SpcR promoter region (the full region and several truncations) to understand their transcriptional strengths.

The SpcR Full Length (FL) promoter was PCR amplified, and this product was used in BsaI GGA (Engler et al., 2008). Our procedures are summarized on this page, while the specific details can be found in the wiki, on the Experiments webpage.

The SpcR FL PCR product, mScarlet RFP reporter, and pBTK300 rpoC terminator were inserted into pIB184, a GFP dropout vector backbone, to produce an assembly plasmid (BBa_K5119050). This assembly plasmid was made with the BsaI NEB Golden Gate Assembly Kit (Potapov, V. et al.). The GGA product was sequenced and confirmed through Plasmidsaurus Whole Plasmid Sequencing.

Following GGA, the SpcR FL assembly plasmid was transformed into chemically competent DH5α cells through a heat shock protocol adapted from (Sambrook, J. and Russell, D.W., 2001). These cells were grown on a recovery plate containing LB media and erythromycin (ERY) antibiotic. A GFP-negative colony was picked from the recovery plate to make a culture for the fluorescence assay. Prior to the fluorescence assay, duplicate cultures of the SpcR FL colony were made. A culture containing only LB media and ERY was the negative control. The positive controls were three Anderson Promoters, BBa_J23100, BBa_J23101, and BBa_J23114. All cultures were incubated at 37°C for 24 hours. Right before incubation, calibration beads from the iGEM Measurement toolkit were prepared on a 96-well plate following the Interlab protocol (Baldwin et al., 2019; Beal et al., 2020). Post-incubation, 100 µL of each culture was pipetted on the 96-well plate. The plate reader OD was set to 660 for the fluorescence assay, and the excitation and emissions wavelengths were set to 561 nm and 610 nm, respectively. This assay was repeated three times on separate days to ensure consistency in the results.

Characterization

Figure 4: Recovery Plate for transformed SpcR FL DH5-α cells. The recovery plate contains LB-media and erythromycin antibiotic. Successfully transformed colonies are GFP-negative.

The DH5-α cells were transformed with composite part BBa_K5119050. This composite part has a GFP dropout vector substituted by the insert [SpcR FL + mScarlet RFP reporter + rpoC terminator]. Hence, any GFP-negative colonies were likely to be successful transformations. Waiting for graph from Annie
Figure 5: RFP fluorescence assay for all antibiotic resistance promoter constructs. Blue and orange represent the first and second of each pair of duplicates respectively. Transcriptional strength is quantified in terms of Fluorescence/CFU averaged over three trials.
The pBTK1028 [...], pBTK1029 [BBa_J23100], pBTK1030 [BBa_J23101], and pBTK1039 [BBa_J23114] promoters were positive controls with known transcriptional strengths. The average Fluorescence/CFU value for SpcR FL is low relative to the positive controls. This implies that the SpcR FL promoter has a low transcriptional strength and may contribute weakly to the upregulation of gene expression.

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. <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 /li>
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