Part:BBa_K5119017
SpcR FL Promoter
This part is a full-length spectinomycin antibiotic resistance promoter originally found in Streptomyces spectabilis 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.
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.
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
- Enter Registry Name of Composite part – 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 theProject Description. Experiments webpage of the UT Austin 2024 iGEM Wiki.
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. Three Anderson Promoters, BBa_J23100, BBa_J23101, and BBa_J23114, were the positive controls. 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. For the fluorescence assay, the plate reader OD was set to 660, 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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
| None |