DOI: https://doi.org/10.15368/theses.2010.61
Available at: https://digitalcommons.calpoly.edu/theses/297
Date of Award
5-2010
Degree Name
MS in Biological Sciences
Department/Program
Biological Sciences
Advisor
Michael Black
Abstract
Celiac disease (CD) is an autoimmune disorder that affects approximately 1% of the population [55]. CD is characterized by intestinal villus atrophy after consumption of gluten from wheat, barley, or rye. Patients with CD often experience abdominal pain, diarrhea, malnutrition, fatigue, and a failure to thrive. There is currently no treatment for CD. Patients must live on a strict lifelong exclusion of dietary gluten. Due to the high content of gluten in western diets and poor labeling of gluten content, adherence to a gluten free diet (GFD) is difficult [15].Nearly all the enzymes that can digest the gluten peptide are sensitive to the stomach's low pH . As a result, dietary supplementation with enzymes to digest gluten has yet to produce a viable alternative treatment to a GFD.
We propose to use a resident microbe of the human intestinal tract to express a peptidase to digest the immunoreactive gluten fragments. The bacteria, L. reuteri, will colonize the host's intestines and digest the gluten peptides before causing an autoimmune response. To accomplish this task, this thesis describes a food grade, plasmid based system to integrate genes into the genome of L. reuteri. The plasmid system utilizes an origin of replication that requires a protein, RepA, to propagate itself. A helper plasmid provides the RepA protein in trans to an integration plasmid that cannot provide RepA to itself. The integration plasmid carries a homologous region to the genome of L. reuteri allowing for targeted genomic integration. The integration plasmid will not replicate on its own, and will be integrated into the genome if the helper plasmid is absent. To select for these genomic integrants the integration plasmid expresses an erythromycin resistance marker. Using the Cre/Lox system the antibiotic resistance will be removed from the bacterial genome to re-establish the L. reuteri's food grade status. This thesis describes the construction and verification of the above mentioned plasmid tool kit containing the helper, integration, and Cre expression plasmids to integrate genes into the L. reuteri genome.