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1. STAPHYLOCIDE: Delivering Antibiotic Resistance Gene
Silencing Mechanisms to a MRSA Population using Bacterial
Conjugation
2. "The problem is so serious that it threatens the
achievements of modern medicine. - World Health Organization,
Antimicrobial Resistance: Global Report on Surveillance 2014
3. 80 461
4. 11 285
5. Infectious Diseases Society of America Clin Infect Dis.
2011; 52:S397-S428Adapted from: Data collected from hospital
intensive care units that participate in the National Nosocomial
Infections Surveillance System of the Centers for Disease Control.
MRSA Cases by Year 16 14 12 7 4 2 0 2 4 6 8 10 12 14 16 18 Number
of new antimicrobial agents approved by the FDA for humans
CasesinThousands
6. MRSA resistance in a nutshell Penicillin PBP Chromosome Cell
Wall Staphylococcus aureus
7. MRSA resistance in a nutshell Methicillin Resistant
Staphylococcus aureus mecA gene Penicillin PBP2A Chromosome Cell
Wall
8. mecA mRNA Transcription Translation PBP2 A mecA mRNA
Transcription Translation PBP2 A mecA mRNA Transcription
Translation PBP2 A MRSA STAPHYLOCIDE
9. IMPROVING THE REGISTRY
10. 9 Promoters 1. sarA P1 Strong constitutive 2. Xylose
inducible promoter construct Ribosome Binding Sites 1. sodA RBS 2.
Optimized TIR RBS Terminators 1. sarA rho-independent
Staphylococcal Parts Selection Markers 1. ermM Erythromycin
resistance 2. aadD Kanamycin resistance 3. spC Spectinomycin
resistance Origin of Replication 1. pSK41 - S. aureus - Theta
Replictation - Low copy Reporters - DsRed - YFP
11. 10 S. epidermidis (ATCC 12228) Level 1 organism Native to
human microbiota Able to conjugate with S. aureus No endogenous
CRISPR system unlike other S. epidermidis strains Staphylococcal
Strain
12. 11 Reporter Gene: DsRed E. coli S. epidermidis -ve
control
13. 12 E. coli-Staphylococcus Shuttle Vector BBa_K1323017
ErmRoriVE. coliCmR oriVS.aureus P SRFP Expression Cassette
(BBa_J04450) VF2 VR Improved pSB1C3 by making it more versatile:
pSB1C3 parts Parts we introduced
14. 13 Shuttle Vector: Antibiotic Resistance Stably maintained
in S. epidermidis Confers erythromycin resistance
31. 30 Silence: RNAi Preliminary Test YFP Alone Control sRNA1
sRNA2 sRNA3 RFU/OD600
32. 31 Characterize silencing systems in S. epidermidis
Integrate yfp into S. epidermidis genome Incorporate the mecA gene
regulation Silence: Future Directions
33. DELIVE R ModelingLab Design
34. 33 Conjugation in Staphylococcus Solid Surface
RecipientDonor
35. 34 Deliver: Conjugation Advantages: Large carrying capacity
Independently propagates Opportunity to contribute to an
underdeveloped area of research Disadvantage: Not efficient
36. 35 Conjugation Parts: pGO1 pGO1: S. aureus conjugational
plasmid oriT-nes: BBa_K1323003 oriT nesRBS TT 2.2 kb trs Region:
Still in progress trs: 13.5 kb
37. 36 Conjugation Test Construct pSBS1A3 ErmR AmpRoriVE.coli P
DsRed TTRBS oriVS. aureusnes TTRBSoriTtrs genes S RecipientsDonor
Transconjugants Filter Mating Assays
38. 37 Deliver: Modeling Challenge: Modeling conjugation
between cells spread across a lab plate or a patients skin
39. 38 Deliver: Modeling Two novel models: Partial Differential
Equation (PDE) is deterministic and computationally efficient
Agent-Based Approach is stochastic and considers the spatial
relationships between individual cells Output: time needed for
silencing to spread
40. 39 Deliver: Agent Based Model Staphylococcus conjugation
rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t
= 0 ht = 0 h
41. 40 Deliver: Agent Based Model Staphylococcus conjugation
rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t
= 6 h t = 6 h
42. 41 Deliver: Agent Based Model Staphylococcus conjugation
rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t
= 12 h t = 12 h
43. 42 Deliver: Agent Based Model Staphylococcus conjugation
rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t
= 24 h t = 24 h
44. 43 Deliver: Agent Based Results
45. 44 Deliver: PDE Model Results
46. 45 Deliver: Future Uses of Model + Find igem-waterloo on
GitHub!
47. 46 Improve conjugation efficiency with error prone PCR
mutagenesis and selective mating assays Deliver: Future Directions
Test conjugational efficiency in S. epidermidis
48. TRANSLAT E AdaptabilitySafetyMarket Viability
49. 48 Translate: Commercialization STAPHYLOCIDE Plasmid
Conjugation Parts
56. 55 Submitted 19 BioBricks, 8 characterized Improved
BioBrick backbone to develop shuttle vector Produced and validated
several models of the silencing and delivery systems Explored
scalability of project Collaborated on uOttawa iGEM & Virginia
Tech project and assisted with oGEM Accomplishments
57. 56 Accomplishments: Outreach High School Enrichment
ProgramScience Club Lab Skills Video Series Sir John A. Macdonald
Secondary School
58. 57 Acknowledgements Dr. Marc Aucoin Dr. Brian Ingalls Dr.
Matthew Scott Dr. Trevor Charles Dr. Barbara Moffat Dr. Andrew
Doxey
59. Questions?
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