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