Results

Introduction

Our team's wet-lab journey began in rather unorthodox fashion. While we were still in the ideation phase for our project goals, our first results in lab came from a small-scale project centered on characterizing a catalogue of broad-host range promoters derived from antibiotic resistance promoter regions. This project would become a cornerstone for our future work, serving as an introduction to synthetic biology bioengineering for many of us in addition to providing us with an array of promoter parts we would later use in several of our constructs. Once we had an established a project goal, our promoter project took a backseat to our other project objectives. However, it remained a significant aspect of our wet lab work and comprised one of the four project areas which subsequently formed around our developed project goal. Our primary project goal which is the secretion of gliadin-degrading enzymes from a model bacterial organism, consisted of the following 4 experimental objectives:

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