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Introduction

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Image 1

Figure 1. How each objective contributes to our final modular system and chassis. Created with Biorender.com

To develop an engineered probiotic bacterium that could degrade trace amounts of gluten for people with celiac disease, we adopted a collaborative research strategy that involved exploring four primary objectives:

  1. Characterize several antibiotic resistance gene promoters and test their strength in E. coli.
    1. Succeeded in characterizing kanamycin and ampicillin full length and variant promoters, which were used for several constructs in other parts of the project
  2. Measure the efficiency of SecB signal peptides in secreting fluorescent proteins to the extracellular milieu.
    1. Encountered several challenges secreting fluorescent proteins from the inducible pET28a plasmid using the T7 promoter and ultimately transitioned to pIB184-GFP. Learned that signal peptides seem to work better under lower strength promoters
    2. PelB and USP45TM8 appeared to be the best performing tags in E. coli overall for SecB based secretion.
    3. USP45TM8 mutated to form a potentially novel signal peptide sequence for secretion.
  3. Transform GI-tract associated gram-positive bacteria with modular plasmids and run assays to test our final modular system.
    1. Domesticated and improved modularity of broad-host range expression plasmids pIB184-GFP and pIB165-GFP for various assemblies.
    2. Transformations into proposed gram-positive bacteria were attempted.
    3. Attempted creating assembly plasmids containing known gliadin-degrading enzyme coding sequences.
    4. Successfully electroporated secretion-based and standard fluorescence constructs into L. lactis.
  4. Create, optimize, and test various degradative assays to assess bacterial proteolytic activity along with assessing bacterial viability in simulated gastric conditions.
    1. Isolated three species from kimchi and four from sourdough starter capable of degrading gluten; not all gluten-degrading strains targeted gliadin
    2. Succesfully observed gliadin degradation using gliadin-infused agar plates in 6 bacterial species