Jamboree final presentation!

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<ol><li> 1. STAPHYLOCIDE: Delivering Antibiotic Resistance Gene Silencing Mechanisms to a MRSA Population using Bacterial Conjugation </li><li> 2. "The problem is so serious that it threatens the achievements of modern medicine. - World Health Organization, Antimicrobial Resistance: Global Report on Surveillance 2014 </li><li> 3. 80 461 </li><li> 4. 11 285 </li><li> 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 </li><li> 6. MRSA resistance in a nutshell Penicillin PBP Chromosome Cell Wall Staphylococcus aureus </li><li> 7. MRSA resistance in a nutshell Methicillin Resistant Staphylococcus aureus mecA gene Penicillin PBP2A Chromosome Cell Wall </li><li> 8. mecA mRNA Transcription Translation PBP2 A mecA mRNA Transcription Translation PBP2 A mecA mRNA Transcription Translation PBP2 A MRSA STAPHYLOCIDE </li><li> 9. IMPROVING THE REGISTRY </li><li> 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 </li><li> 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 </li><li> 12. 11 Reporter Gene: DsRed E. coli S. epidermidis -ve control </li><li> 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 </li><li> 14. 13 Shuttle Vector: Antibiotic Resistance Stably maintained in S. epidermidis Confers erythromycin resistance </li><li> 15. DELIVERSILENC E TRANSLATE </li><li> 16. SILENC E DesignTranslationTranscription </li><li> 17. 16 Silence YFP mRNA Transcription Translation YFP </li><li> 18. 17 Silence: CRISPRi dCas9-sgRNA complex blocks RNA polymerase </li><li> 19. 18 Silence: CRISPRi Network </li><li> 20. 19 Silence: CRISPRi Results </li><li> 21. 20 Silence: CRISPRi Sensitivity Sensitive to mRNA degradation rate Therefore Targeting of translation will improve silencing! </li><li> 22. 21 Silence: RNAi sRNA-Hfq complex blocks ribosome </li><li> 23. 22 Silence: RNAi Network </li><li> 24. 23 Silence: RNAi Results </li><li> 25. 24 Silence: Design YFP mRNA Transcription Translation YFP CRISPRi dCas9-sgRNA Complex </li><li> 26. 25 pSB1A3 AmpRoriVE.coli ErmR oriVS. aureus TTsgRNA Pconst Silence: CRISPRi dCas9 xylose XylR TT </li><li> 27. 26 Silence: Design YFP mRNA Transcription Translation YFP RNAi CRISPRi dCas9-sgRNA Complex Hfq-sRNA Complex </li><li> 28. 27 pSB1A3 AmpRoriVE.coli ErmR oriVS. aureus TTsRNA Pconst Silence: RNAi Hfq xylose TTxylR </li><li> 29. 28 Silence: RNAi Design YFP CDSScarRBS YFP mRNA 5 3 sRNA 1 sRNA 2 sRNA 3 </li><li> 30. 29 Co- Transform Silence: RNAi Preliminary Tests pSB3K3 E. coli DH5 Measure fluorescence pSB1A3 </li><li> 31. 30 Silence: RNAi Preliminary Test YFP Alone Control sRNA1 sRNA2 sRNA3 RFU/OD600 </li><li> 32. 31 Characterize silencing systems in S. epidermidis Integrate yfp into S. epidermidis genome Incorporate the mecA gene regulation Silence: Future Directions </li><li> 33. DELIVE R ModelingLab Design </li><li> 34. 33 Conjugation in Staphylococcus Solid Surface RecipientDonor </li><li> 35. 34 Deliver: Conjugation Advantages: Large carrying capacity Independently propagates Opportunity to contribute to an underdeveloped area of research Disadvantage: Not efficient </li><li> 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 </li><li> 37. 36 Conjugation Test Construct pSBS1A3 ErmR AmpRoriVE.coli P DsRed TTRBS oriVS. aureusnes TTRBSoriTtrs genes S RecipientsDonor Transconjugants Filter Mating Assays </li><li> 38. 37 Deliver: Modeling Challenge: Modeling conjugation between cells spread across a lab plate or a patients skin </li><li> 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 </li><li> 40. 39 Deliver: Agent Based Model Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 0 ht = 0 h </li><li> 41. 40 Deliver: Agent Based Model Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 6 h t = 6 h </li><li> 42. 41 Deliver: Agent Based Model Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 12 h t = 12 h </li><li> 43. 42 Deliver: Agent Based Model Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 24 h t = 24 h </li><li> 44. 43 Deliver: Agent Based Results </li><li> 45. 44 Deliver: PDE Model Results </li><li> 46. 45 Deliver: Future Uses of Model + Find igem-waterloo on GitHub! </li><li> 47. 46 Improve conjugation efficiency with error prone PCR mutagenesis and selective mating assays Deliver: Future Directions Test conjugational efficiency in S. epidermidis </li><li> 48. TRANSLAT E AdaptabilitySafetyMarket Viability </li><li> 49. 48 Translate: Commercialization STAPHYLOCIDE Plasmid Conjugation Parts </li><li> 50. 49 Translate: Commercialization </li><li> 51. 50 Translate: Commercialization </li><li> 52. 51 Translate: Commercialization -Lactam Antibiotic </li><li> 53. 52 Translate: Commercialization -Lactam Antibiotic </li><li> 54. 53 Translate: Adaptability </li><li> 55. DELIVERSILENC E TRANSLATE </li><li> 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 &amp; Virginia Tech project and assisted with oGEM Accomplishments </li><li> 57. 56 Accomplishments: Outreach High School Enrichment ProgramScience Club Lab Skills Video Series Sir John A. Macdonald Secondary School </li><li> 58. 57 Acknowledgements Dr. Marc Aucoin Dr. Brian Ingalls Dr. Matthew Scott Dr. Trevor Charles Dr. Barbara Moffat Dr. Andrew Doxey </li><li> 59. Questions? </li><li> 60. 59 Bayer, M. G., Heinrichs, J. H., &amp; 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., &amp; 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., &amp; 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., &amp; 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 </li><li> 61. 60 Malone, C. L., Boles, B. R., Lauderdale, K. J., Thoendel, M., Kavanaugh, J. S., &amp; 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., &amp; 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., &amp; 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 </li></ol>