1. 1. STAPHYLOCIDE: Delivering Antibiotic Resistance Gene Silencing Mechanisms to a MRSA Population using Bacterial Conjugation
2. 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. 3. 80 461
4. 4. 11 285
5. 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. 6. MRSA resistance in a nutshell Penicillin PBP Chromosome Cell Wall Staphylococcus aureus
7. 7. MRSA resistance in a nutshell Methicillin Resistant Staphylococcus aureus mecA gene Penicillin PBP2A Chromosome Cell Wall
8. 8. mecA mRNA Transcription Translation PBP2 A mecA mRNA Transcription Translation PBP2 A mecA mRNA Transcription Translation PBP2 A MRSA STAPHYLOCIDE
9. 9. IMPROVING THE REGISTRY
10. 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. 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. 12. 11 Reporter Gene: DsRed E. coli S. epidermidis -ve control
13. 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. 14. 13 Shuttle Vector: Antibiotic Resistance Stably maintained in S. epidermidis Confers erythromycin resistance
15. 15. DELIVERSILENC E TRANSLATE
16. 16. SILENC E DesignTranslationTranscription
17. 17. 16 Silence YFP mRNA Transcription Translation YFP
18. 18. 17 Silence: CRISPRi dCas9-sgRNA complex blocks RNA polymerase
19. 19. 18 Silence: CRISPRi Network
20. 20. 19 Silence: CRISPRi Results
21. 21. 20 Silence: CRISPRi Sensitivity Sensitive to mRNA degradation rate Therefore Targeting of translation will improve silencing!
22. 22. 21 Silence: RNAi sRNA-Hfq complex blocks ribosome
23. 23. 22 Silence: RNAi Network
24. 24. 23 Silence: RNAi Results
25. 25. 24 Silence: Design YFP mRNA Transcription Translation YFP CRISPRi dCas9-sgRNA Complex
26. 26. 25 pSB1A3 AmpRoriVE.coli ErmR oriVS. aureus TTsgRNA Pconst Silence: CRISPRi dCas9 xylose XylR TT
27. 27. 26 Silence: Design YFP mRNA Transcription Translation YFP RNAi CRISPRi dCas9-sgRNA Complex Hfq-sRNA Complex
28. 28. 27 pSB1A3 AmpRoriVE.coli ErmR oriVS. aureus TTsRNA Pconst Silence: RNAi Hfq xylose TTxylR
29. 29. 28 Silence: RNAi Design YFP CDSScarRBS YFP mRNA 5 3 sRNA 1 sRNA 2 sRNA 3
30. 30. 29 Co- Transform Silence: RNAi Preliminary Tests pSB3K3 E. coli DH5 Measure fluorescence pSB1A3
31. 31. 30 Silence: RNAi Preliminary Test YFP Alone Control sRNA1 sRNA2 sRNA3 RFU/OD600
32. 32. 31 Characterize silencing systems in S. epidermidis Integrate yfp into S. epidermidis genome Incorporate the mecA gene regulation Silence: Future Directions
33. 33. DELIVE R ModelingLab Design
34. 34. 33 Conjugation in Staphylococcus Solid Surface RecipientDonor
35. 35. 34 Deliver: Conjugation Advantages: Large carrying capacity Independently propagates Opportunity to contribute to an underdeveloped area of research Disadvantage: Not efficient
36. 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. 37. 36 Conjugation Test Construct pSBS1A3 ErmR AmpRoriVE.coli P DsRed TTRBS oriVS. aureusnes TTRBSoriTtrs genes S RecipientsDonor Transconjugants Filter Mating Assays
38. 38. 37 Deliver: Modeling Challenge: Modeling conjugation between cells spread across a lab plate or a patients skin
39. 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. 40. 39 Deliver: Agent Based Model Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 0 ht = 0 h
41. 41. 40 Deliver: Agent Based Model Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 6 h t = 6 h
42. 42. 41 Deliver: Agent Based Model Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 12 h t = 12 h
43. 43. 42 Deliver: Agent Based Model Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 24 h t = 24 h
44. 44. 43 Deliver: Agent Based Results
45. 45. 44 Deliver: PDE Model Results
46. 46. 45 Deliver: Future Uses of Model + Find igem-waterloo on GitHub!
47. 47. 46 Improve conjugation efficiency with error prone PCR mutagenesis and selective mating assays Deliver: Future Directions Test conjugational efficiency in S. epidermidis
48. 48. TRANSLAT E AdaptabilitySafetyMarket Viability
49. 49. 48 Translate: Commercialization STAPHYLOCIDE Plasmid Conjugation Parts
50. 50. 49 Translate: Commercialization
51. 51. 50 Translate: Commercialization
52. 52. 51 Translate: Commercialization -Lactam Antibiotic
53. 53. 52 Translate: Commercialization -Lactam Antibiotic
54. 54. 53 Translate: Adaptability
55. 55. DELIVERSILENC E TRANSLATE
56. 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. 57. 56 Accomplishments: Outreach High School Enrichment ProgramScience Club Lab Skills Video Series Sir John A. Macdonald Secondary School
58. 58. 57 Acknowledgements Dr. Marc Aucoin Dr. Brian Ingalls Dr. Matthew Scott Dr. Trevor Charles Dr. Barbara Moffat Dr. Andrew Doxey
59. 59. Questions?
60. 60. 59 Bayer, M. G., Heinrichs, J. H., & Cheung, A. L. (1996). The molecular architecture of the sar locus in Staphylococcus aureus. Journal of Bacteriology, 178(15): 4563-70 Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. a. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research, 41(15): 74293 Bose, J. L., Fey, P. D., & Bayles, K. W. (2013). Genetic Tools to Enhance the Study of Gene Function and Regulation in Staphylococcus aureus. Applied Environmental Microbiology, 79(7): 2218- 2224. Caryl, J. A. and ONeill, A. J. (2009). Complete nucleotide sequence of pGO1, the prototype conjugative plasmid from the staphylococci. Plasmid, 62: 35-38 Cirino, P. C., Mayer, K. M., and Umeno, D. (2002). Chapter 1: Generating Mutant Libraries Using Error-Prone PCR, Methods in Molecular Biology, vol. 231. New Jersey: Humana Press Inc. Climo, M. W., Sharma, V. K., and Archer, G. L. (1996). Identification and Characterization of the Origin of Conjugative Transfer (oriT) and a Gene (nes) Encoding a Single-Stranded Endonuclease on the Staphylococcal Plasmid pGO1. Journal of Bacteriology, 178 (16): 4975-83 Fey, P. D. (2014). Staphylococcus epidermidis: methods and protocols. New York: Springer Science + Business Media, LLC. Horstmann, N., Orans, J., Valentin-Hansen, P., Shelburne III, S. A., & Brennan, R. G. (2012). Structural mechanism of Staphylococcus aureus Hfq binding to an RNA A-tract. Nucleic Acids Research, 1-13. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012). A Programmable Dual-RNAGuided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337: 816-821. Katze, M. J., He, Y., and Gale, M. (2002). Viruses and Interferon: A Fight for Supremacy. Nature Reviews, 2: 675-687. Larson, M. H., Gilbert, L. A., Wang, X., Lim, W. A., Weissman, J. S., and Qi, L. S. (2013). Nature Protocols, 8 (11): 2180-2196. References
61. 61. 60 Malone, C. L., Boles, B. R., Lauderdale, K. J., Thoendel, M., Kavanaugh, J. S., & Horswill, A. R. (2009). Fluorescent Reporters for Staphylococcus aureus. Journal of Microbiological Methods, 77(3): 251-260. Qi, L. S., Larson, M. H., Gilbert, L. a, Doudna, J. a, Weissman, J. S., Arkin, A. P., & Lim, W. a. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5): 117383. Yoo, S. M., Na, D., & Lee, S. Y. (2013). Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nature Protocol, 8 (9): 1694-1707. Zhang, Y-Q. (2003). Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228). Molecular Microbiology, 49(6): 1577-1593. Zhao, H. (2004). Staggered Extension Process In Vitro DNA Recombination, Methods in Enzymology, vol. 388, 42-49. References