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. 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.

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.

1. Enter Registry Name of Composite part – BBa_K5119050

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]