P. acnes Exploring Our Skin Engineering P. acnes Skin Probiotics Computer Validation Electroporation Cathelicidin Melanin Desaturase Boolean Network Simulation Pitt iGEM 2014 Team DermGerm: Kita S 1 , Sawlani S 1 , Waters S 1 , Freer D 1 , Dash S 2 , Jacus M 1 , Huang W 2 , Campbell K 2 , Padinjarekutt S 3 1 Dept. of Bioengineering, 2 Dept. of Biology, 3 Dept. of Chemistry University of Pittsburgh, Pittsburgh, PA Boolean Network Model • Topical cathelicidin via bacteria is a novel treatment for acne-related inflammation. • However, typical modeling is inadequate for large volume of skin interactions. • Boolean network provides simplification. • Biologic circuits == digital circuits! Network Model • Network diagram of cathelicidin interaction in the skin, showing feed-forward control. Human Practices – Educate Human Practices – Engage Pitt Science Outreach • SciencePalooza 2014 – Presented “Dermalicious,” a module about skin care. • Taught to middle school children with 100% satisfaction rating. • Tailgate – Presented “Enzyme Time,” a module demonstrating the action & importance of enzymes in biology at Pitt football game. Duquesne University Ethics Forum • Presented case study of Dr. Albert Kligman and Holmesburg Prison Experiments. • Promoted and encouraged the study of ethics, especially as it pertains to synthetic biology. Pitt iGEM Blog • Tips and tricks for first year iGEM teams. • 322 page views from 12 countries. • Visit us at: http ://igempitt.wordpress.com/ Crowdfunding Campaign • Organized campaign using Experiment.com. • 108% funded with $2063 raised. • 1,773 page views from 155 sources. Cathelicidin • Natural antibiotic produced by immune cells. • Constructed BioBrick combines cathelicidin with blue light induction system. • YF1 sensor → FixJ regulator → FixK1 promoter. • Cathelicidin only expressed under blue light. • Submitted BioBrick systems: 1. Blue Light Promoter (FixJ) → (RBS) → mRFP1 → HSP60 Promoter → Blue Light Sensor (YF1/FixJ) 2. Blue Light Promoter (FixJ) → (RBS) → mRFP1 → Cathelicidin • Complete BioBrick system: • Blue Light Promoter (FixJ) → (RBS) → mRFP1 → Cathelicidin → Terminator → HSP60 Promoter → Blue Light Sensor (YF1/FixJ) Accomplishments Five BioBrick parts submitted: 1. HSP60 promoter from M. bovis (BBa_K1548000) 2. HSP60 promoter + TM4 phage RBS (BBa_K1548001) 3. HSP60 + mRFP1 (BBa_K1548002) 4. HSP60 + blue light regulator + mRFP1 (BBa_K1548003) 5. HSP60 + blue light regulator + mRFP1 + cathelicidin (BBa_K1548004) Characterized submission # 4. in E. coli. Validated cathelicidin treatment via simulations. Educated over 100 children about skin health. Collaborated with several first-year iGEM teams. • In-person and how-to articles on our blog. Simulation Results • Ratio of inflammation due to the immune system over inflammation due to injury. Topical Cathelicidin scenario shows less inflammation due to injury, which promotes healthier skin. • Average value of injury over time. Topical Cathelicidin scenario shows less inflammation in between cycles of inflammation. Electroporation Protocol Bacteria Growth 1. Grow up desired p. acnes strain 30 mL of A media until optical density (OD) of 0.5. 2. Add glycine then grow bacteria overnight. Electroporation 8. Thaw competent cells. Add plasmid DNA and TypeOne RME inhibitor. 9. Put mixture in cuvette and cuvette into machine. Pulse. Competent Cells 3. Centrifuge p. acnes with 10% glycerol buffer. 4. Soak in 15% glycerol buffer with lysozyme for 2 hours. 5. Centrifuge with buffer. 6. Freeze with dry ice in 50 uL aliquots of 10% glycerol buffer. Recovery 10. Directly after electroporation, place cells in 500 uL of media overnight. 11. Streak bacteria onto plates with erythromycin. 12. After a week of growth on the plates, count and validate colonies. Desaturase • Increased P. acnes on skin is correlated with lower concentrations of linoleic acid and acne-related inflammation. • Δ12-Fatty Acid Desaturase (Δ12-FAD) restores linoleic acid. • By expressing Δ12-FAD in P. acnes, we hope to reduce inflammation by rebalancing skin oils. • HSP60 Promoter → (RBS) → mRFP1 → Desaturase → Terminator. Δ12-FAD Linoleic Acid Oleic Acid K1027002 Attributions • Funding o Experiment.com o Pitt – Dept. Biology, Bioengineering Drs. Grabowski, Shroff, Davidson, Patzer • Mentors o Hatfull Lab Carlos Guerrero, Ching-Chung Ko o Jason Lohmueller o Natasa Miskov-Zivanov o Joseph Ayoob • Graphic Team o Melissa Thompson o Anand Mahalingam o Catherine Kita o Ryan Black • Outreach o Amie DiTomasso o Sak Kia Goh o Andrew Falk Design of Experiments • Electroporation transformation efficiency is affected by dozens of variables. • Statistical design of experiments (DOX) offers efficient testing of many variables at once using ANOVA. • P. acnes strain • Culture Temp (C) • Glycine (%) • Lysozyme (mg/mL) • Plasmid DNA (ug) • E Field (kV/cm) • Incubation Temp (C) • Restriction Enzymes Skin Probiotics • Skin probiotics are a developing field, which offers promising treatment for skin disease (like acne). • Genetically engineering a skin bacterium provides a way to engineer novel functionalities to human skin without engineering human cells. Engineered P. acnes skin probiotic • Propionibacterium acnes (P. acnes) is a commensal skin bacterium found on major skin areas (face,back neck, arms, and legs). • Genetic research on P. acnes is scarce because no effective transformation protocol currently exists. Melanin • Responsible for skin pigment • Natural mechanism to protect cells from UV radiation • Double digestion of shipment plasmid containing melanin part with XbaI and PstI • The plasmid was not confirmed to be up to BioBrick standard potentially causing a negative result. Melanin and Vector Backbone Protocol Results 1 No growth after electroporation. 2 Growth resembling P. acnes, but failed PCR validation. 3 Growth does not resemble P. acnes. Future Directions • Transition into other skin bacteria strains. Phylum: Firmicutes Species: Staphylococcus aureus Relevant locations: Occiput, Glabella, Manubrium Protocol: Monk et. Al, 2012 Phylum: Firmicutes Species: Staphylococcus epidermidis Relevant locations: Occiput, Glabella, Manubrium Protocol: Monk et. Al, 2012 Phylum: Actinobacteria Species: Micrococciaceae lysodeikticus Relevant locations: Occiput, back Protocol: Kloos, 1969