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P. acnesExploring
Our Skin
Engineering
P. acnes
Skin
Probiotics
Computer
Validation
Electroporation
Cathelicidin
MelaninDesaturase
Boolean
Network
Simulation
Pitt iGEM 2014Team DermGerm:Kita S1, Sawlani S1, Waters S1, Freer D1, Dash S2, Jacus M1, Huang W2, Campbell K2, Padinjarekutt S3
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)
AccomplishmentsFive 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 ProtocolBacteria Growth1. 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.
Electroporation8. Thaw competent cells. Add plasmid
DNA and TypeOne RME inhibitor.
9. Put mixture in cuvette and
cuvette into machine. Pulse.
Competent Cells3. 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-FADLinoleic AcidOleic Acid
K1027002
Attributions• Funding
o Experiment.com
o Pitt – Dept. Biology, Bioengineering Drs. Grabowski, Shroff, Davidson, Patzer
• Mentorso Hatfull Lab
Carlos Guerrero, Ching-Chung Ko
o Jason Lohmueller
o Natasa Miskov-Zivanov
o Joseph Ayoob
• Graphic Teamo Melissa Thompson
o Anand Mahalingam
o Catherine Kita
o Ryan Black
• Outreacho 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 Results1 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
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