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Designed an expression system for protein secretion in Lactobacillus. Built a signal sequence and promoter for Lactobacillus species bacteria. Constructed the DNA insert containing peptide p1025, ready for expression. Successfully transformed Lactobacillus bulgaricus and Lactobacillus lactis. Electrotransformed with plasmids pJK650 and pLEM415. Determined plasmid pTG262 unsuitable for electroporation into Lactobacillus. Successfully developed a novel binding assay to evaluate p1025. DNA sequence constructed to be expressed with multiple tags to facilitate testing. SUMMARY As proof of concept, engineer yogurt bacteria to prevent dental cavities. Streptococcus mutans is a leading cause of dental caries. p1025 (20aa) competitively inhibits binding of S. mutans – harmless bacteria grow in its place. (Kelly et al.; Nature Biotechnol. 1999) Engineer yogurt bacteria Lactobacillus to secrete this peptide under control of a lactose activated promoter. Develop expression system and optimal protocols for transforming Lactobacillus. Develop novel binding assay to characterize the effectiveness of p1025. Expand scope of the registry to include parts for lactic acid bacteria. DNA CONSTRUCT RESULTS MOTIVATION Large barriers to drug delivery in underdeveloped regions. Cumbersome infrastructure needed to support supply chains. Shorten supply chain of drug delivery by engineering probiotic yogurt. Sustainable and efficient on site drug and nutrient production. Self-sustainable supply of BiOGURT BINDING ASSAY Experiment showing effect of saliva on S. mutans binding Why is this assay novel? Added a de-chaining step for S. mutans. Coated hydroxyapatite beads with BSA to prevent non-specific binding. Increased saliva coating time. Washed beads and then plated to confirm S. mutans attaches to beads. Serial dilution of supernatant and spotting of all dilutions. p1025 Expression in E. coli System Diagram TEAM CONTRIBUTIONS BioBrick Parts: Lactobacillus Parts: Lactobacillus secretion signal sequence Lactobacillus LacS native promoter pLEM415 plasmid with BioBrick insert pJK650 plasmid with BioBrick insert p1025 peptide to prevent tooth decay Developed Protocols: Lactobacillus electroporation protocol Culturing Lactobacillus and other lactic acid bacteria Miniprep protocol for Lactobacillus Tooth binding assay for p1025 FUTURE DIRECTIONS Future Goals Further testing Lactobacillus secretion system. Peptide efficiency in yogurt. Chromosomal integration. Remove antibiotic resistance marker. References: Kelly et al. (NatureBiotechnol 1999), Serror et al. (App Env Microbio 2002), Lapierre et al. (Bacteriology 2002), Germond et al. (App Env Microbio 2003) Graduate Advisors: Chia-Yung Wu, Scott Carlson, Lav Varshney, Felix Moser, Vikramaditya Yadav, Woo Chung, Rachel Hilmer, Robbie Barbero, Brian Cook, Laure-Anne Ventouras Faculty Advisors: Drew Endy, Tom Knight Funding: MIT Department of Biological Engineering and MIT UROP office ACKNOWLEDGMENTS PLAN OF ATTACK Construction of p1025 Transform L. bulgaricus Expression of p1025 in E. coli Expression of p1025 in L. bulgaricus Binding assay Make yogurt with transformed L. bulgaricus Parts for fusion protein synthesis: GFP FLAG tag HA tag His tag TEV protease cleavage site Lactobacillus delbruckii Transformation 1. CELL CULTURE 1. Inoculate serial dilutions of fresh bacterial culture. 2. Harvest cultured cells at beginning of stationary phase. 2. WASH BUFFER 1. Wash bacteria with cold electroporation buffer. 3. THERMAL SHOCK 4. ELECTRICAL PULSE 1. Mix cell suspension with compatible plasmid DNA. 2. Subject sample to electric pulse. 5. EXPRESSION 1. Immediately add milk medium or MRS. 6. PLATING, SELECTION 1. Incubate cells, plate on antibiotic MRS plates. 2. Incubate at 37°C for 2-3 days under anaerobic conditions. Lactobacillus delbruckii subsp. bulgaricus Long-term Potential Insertion of other proteins. Pharmaceuticals e.g. anti-Malaria drugs Vitamins and nutrients e.g. Vitamin A Benefits of BiOGURT Engineered bacteria transferred in creation of new batch. Only need one batch to create unlimited supply - elimination of supply chain. Minimal production cost and little training needed. SAFETY Ensure that the peptide is completely secreted. Engineer bacteria to use markers other than resistance to antibiotics. Clinical evaluations of the safety of the product required. Proper dosage needs to be determined. p1025 inhibits binding of S. mutans to saliva coated hydroxyapatite n=2 for no peptide control n=3 for peptide samples error Bar = SD BiOGURT A Self Sustaining System for Nutrient and Vaccine Delivery Andrew Ang, Prarthna Desai, Derek Ju, John Kucharczyk, Asad Moten, Sara Mouradian, Allin Resposo Massachusetts Institute of Technology With saliva Without saliva De-chain S. mutans by passing through syringe. Prepare saliva- BSA coated HA beads with peptides. Add S. mutans to HA beads and extract supernatant after 5 min & 2 HR. Serial dilute supernatant and plate.
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