Team DermGerm Freer D 1 , Jacus M 1 , Kita S 1 , Sawlani S 1 , Waters - - PowerPoint PPT Presentation

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Team DermGerm Freer D 1 , Jacus M 1 , Kita S 1 , Sawlani S 1 , Waters - - PowerPoint PPT Presentation

Feb 24, 2024 607 likes •1.16k views

Team DermGerm Freer D 1 , Jacus M 1 , Kita S 1 , Sawlani S 1 , Waters S 1 , Campbell K 2 , Dash S 2 , Huang W 2 , Padinjarekutt S 3 1 Dept. of Bioengineering, 2 Dept. of Biology, 3 Dept. of Chemistry University of Pittsburgh Im Immunologists Bio

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  • 1Dept. of Bioengineering, 2Dept. of Biology, 3Dept. of Chemistry

University of Pittsburgh

Team DermGerm

Freer D1, Jacus M1, Kita S1, Sawlani S1, Waters S1, Campbell K2, Dash S2, Huang W2, Padinjarekutt S3

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WHO WE ARE

Im Immunologists Derm rmatolo logists Computer Sci cientists Bio ioengineers Bio iolo logists Chemists

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WHO WE ARE

Im Immunologists Derm rmatolo logists Computer Sci cientists

Develo lop Skin in Pro robio ioti tics

Bio ioengineers Bio iolo logists Chemists

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Protect against sun damage Balance skin oils Remove pathogens

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Presence of P. acnes

PROPIONIBACTERIUM ACNES

Commensal skin bacteria associated with acne

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Transformation Probiotic Functions Modeling Human Practices

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Transformation Probiotic Functions Modeling Human Practices

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CHALLENGES

Gram negative  thick cell wall Restriction modification system Growth time - 5 days Anaerobic growth

$100-200 $5

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  • P. ACNES TRANSFORMATION

Goal: Transform P. Acnes using electroporation protocols

Erm

Transformation Protocol:

Cheong et al, 2008

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  • Choose trials carefully for statistical advantage
  • Reveals which parameters have most effect on

successful transformation

Design of Experiments (DOX)

Parameters:

  • P. acnes strain
  • Culture temperature
  • Amount of glycine
  • Lysozyme concentration
  • Restriction enzyme

inhibitor

  • Amount of plasmid DNA
  • Electric field strength
  • Incubation temperature

16

trials instead

  • f 256
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RESULTS

1 No growth after electroporation. 2 Growth resembling P. acnes, but failed PCR validation. 3 Growth does not resemble P. acnes.

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  • Have not yet been able to

transform any P. acnes

  • Different strains
  • Parameters  extreme

conditions

  • Activity of non-type I

restriction systems

DISCUSSION

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Attempt New Species

  • 1. Broad Skin

Location

  • 2. Transformation

Protocols Available

FUTURE DIRECTIONS

  • S. Aureus
  • S. epidermidis
  • M. lysodeikticus
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Transformation Probiotic Functions Modeling Human Practices

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HSP60 Promoter Melanin Cathelicidin Desaturase

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HSP60 PROMOTER

Strong promoter from Mycobacterium bovis

  • Works well in other

propionibacteria

Figure 1. Gel electrophoresis showing presence of HSP60 after PCR. Lanes 2 - 4 contain hsp60 promoter. Lanes 6 - 7 contain hsp60 promoter with an RBS

1 2 3 4 5 6 7

20 0 kb kb .5 .5 kb kb

Part:BBa_K1548000 Part:BBa_K1548001

RBS mRFP1 Hsp60 mRFP1 Hsp60

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HSP60 PROMOTER

Testing in E. coli Not functional As predicted by BPROM Characterized part to be non-functional as expected

1.56E+05 1.58E+05 1.60E+05 1.62E+05 1.64E+05 1.66E+05 1.68E+05 1.70E+05 1.72E+05 1.74E+05 1.76E+05 1.78E+05 Hsp60-RBS-mRFP1 Hsp60-alo lone

mRF RFP! Fl Fluoresence (a (au) u) Cons nstruct

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MELANIN

Goal: Insert MelA into P. acnes to produce melanin

Part:BBa_K274001 – Cambridge ‘09

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MELANIN

Figure 2. Gel electrophoresis showing melanin digestion with XbaI and PstI Lanes 2 & 4 contain melanin and vector backbone.

Goal: Insert MelA into P. acnes to produce melanin

Part:BBa_K274001 – Cambridge ‘09

1 2 3 4 5

3,000 bp Vector Backbone 2,000 bp Expected MelA

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CATHELICIDIN

Goal: Activate controlled cathelicidin production under blue light

  • Dependent on P.

acnes population

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Figure 3. Gel electrophoresis showing cathelicidin system. Lane 2 contains the Blue Promoter – mRFP1 – Cathelicidin part (~1100 bp).

Part:BBa_K1548002 Part:BBa_K1548003

CATHELICIDIN

1 2 3 4

3 kb 1.5 kb

FixJ YF1 FixK2 Hsp60 Cath T1 YF1

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DESATURASE

Goal: Produce desaturase to restore the natural lipid composition of skin

Δ12 12-FAD

Oleic Acid Linoleic Acid

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DESATURASE

Goal: Produce desaturase to restore the natural lipid composition of skin

Δ12 12-FAD

Oleic Acid Linoleic Acid

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Desaturase RBS mRFP1 Terminator Hsp60

DESATURASE

Desaturase digestion with EcoRI & SpeI 1,000 bp Desaturase Part Desaturase

Vector Backbone

mRFP1 digestion with EcoRI & XbaI 3,000 bp Linearized mRFP1 and Vector Backbone

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FUTURE VALIDATION

MELANIN

  • Visual inspection
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FUTURE VALIDATION

CATHELICIDIN

  • Viability assay

under blue-light

MELANIN

  • Visual inspection
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FUTURE VALIDATION

CATHELICIDIN

  • Viability assay

under blue-light

DESATURASE

  • Colorimetric assay

for oleic acid

MELANIN

  • Visual inspection
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Safety

  • P. acnes naturally occurs deep in human skin
  • Cathelicidin is natural kill switch

SAFETY

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Safety

  • P. acnes naturally occurs deep in human skin
  • Cathelicidin is natural kill switch
  • Could add genetic fence:

SAFETY

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Transformation Probiotic Functions Modeling Human Practices

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CATHELICIDIN MODEL

Cathelicidin

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CATHELICIDIN MODEL

Cathelicidin Bacteriocidal

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CATHELICIDIN MODEL

Cathelicidin Bacteriocidal Pro-Inflammatory

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BOOLEAN NETWORKS

Model Biological Circuits as Digital Circuits

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Network Diagram

Normal Input

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Network Diagram

Normal Input Output Output

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Network Diagram

Topical Application

Cathelicidin Input

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Network Diagram

Output Output

Topical Application

Cathelicidin Input

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Node Norm rmal l Condit itions Topical l Cath theli licidin Cathelicidin 23% 35%

  • P. acnes

44% 37% Immune Response (Inflammation) 21% 28% Injury (Inflammation) 52% 42% Ave vera rage In Infl flammation (Immune + Injury) 37% 37% 35% 35%

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Node Norm rmal l Condit itions Topical l Cath theli licidin Cathelicidin 23% 35%

  • P. acnes

44% 37% Immune Response (Inflammation) 21% 28% Injury (Inflammation) 52% 42% Ave vera rage In Infl flammation (Immune + Injury) 37% 37% 35% 35%

Lower growth

  • f P. acnes

Similar average inflammation

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0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1 3 5 7 9 11 13 15 17 19 21 23 25

Avg State Update Round

Injury Node Average

Normal Conditions Topical Cathelicidin

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Transformation Probiotic Functions Modeling Human Practices

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Ethics Research Forum

  • Dr. Albert Kligman and

Holmesburg Prison Experiments Promoted and encouraged the study

  • f ethics in synthetic biology

EDUCATE

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SciencePalooza “Dermalicious” Mobile Science Lab “Enzyme Time”

EDUCATE

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ENGAGE

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Wordpress Blog: http://igempitt.wordpress.com Professional consults How-to articles Team videos

ENGAGE

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COLLABORATIONS

Montgomery HS Pittsburgh Meetup Virginia Survey

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ACKNOWLEDGEMENTS

Advi visors Jas ason Lo Lohmuelle ler – Te Team Mentor, MVP Graham Hat atfull ll – Principal l In Inve vestigator La Lance Dav avid idson – Clu lub Advi visor Nat atasa Mis iskov-Ziv ivanov - Computer Sim imula latio ion Advi visor Sponsors – Univ iversit ity of f Pit ittsburgh San anjeev Shroff – Chair of Bio ioen engin ineering Dep epartment Pau aula la Grabowski i – Chair of f Bio iolo logy Dep epartment Dav avid Vorp rp – Ass ssociate Dea ean of f Researc rch fo for r Swanson School l of f Engineerin ing Pat atri rick Mull llen – Dea ean of f Undergraduate Research fo for Die ietrich Sch chool l of f Arts an and Sci ciences Sponsors – Oth ther Experiment.com te team am – Crowdfundin ing Platform rm La Laboratory Support Car arlos Guerrero – Graduate Student, Hat atfull Lab Lab Chin ing-Chung Ko Ko – Graduate Stu tudent t Hat atfull ll Lab Lab Sak Kia ia Goh – Graduate Stu tudent, Bio ioengineerin ing Gra raphic ic Art rts Ryan Blac ack - Butt tton Mak aker, r, Extr traordin inaire re Cat atherine Kit ita - Graphic Des esigner r Meli lissa Th Thompson – Graphic Desig igner Anan and Mah ahali lingam – Vid ideographer Admin inistra rativ ive Support – Univ iversity of f Pit ittsburgh John Pat atzer – Bio ioen engineering Professor Alic icia Wel elsh – Bio ioen engineering Admin inistrator Pat atri ricia ia Dea ean – Bio iolo logy Admin inistrator Amie ie DiT iTomasso – Sci cience Outreach Coordinator Nic icki i Zev evola la – CEO & Founder, FutureDerm Joseph Ayo yoob – Program Dir irector, TE TECBio REU Pau aul l Kova vach – Dire irector of f Mark rketing an and Communications Andre rew Fal alk – Exec ecutiv ive Dire irector r of f Corp rporate & & Foundation Rel elations

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La Lane Re Reaction 1 1 kb Marker 2 Plasmid + XbaI 3 Plasmid + PstI 4 Plasmid + SpeI 5 Plasmid +EcoRI 6 Plasmid +SpeI and EcoRI

pBRES36a

1 2 3 4 6 5

10 10 kb kb 3 kb kb 1 1 kb kb

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BUILDING THE MODEL

Simulation

Random Starting Conditions Av Average 1,0 ,000 Tria ials Stochastic Updates Literature Searches Network Diagram Boolean Rules

Construction

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BUILDING THE MODEL

Simulation

Random Starting Conditions Av Average 1,0 ,000 Tria ials Stochastic Updates Literature Searches Network Diagram Boolean Rules

Construction

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BUILDING THE MODEL

Simulation

Random Starting Conditions Av Average 1,0 ,000 Tria ials Stochastic Updates Literature Searches Network Diagram Boolean Rules

Construction