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STAPHYLOCIDE : Delivering Antibiotic Resistance Gene Silencing - PowerPoint PPT Presentation

Apr 23, 2023 •359 likes •985 views

STAPHYLOCIDE : Delivering Antibiotic Resistance Gene Silencing Mechanisms to a MRSA Population using Bacterial Conjugation "The problem is so serious that it threatens the achievements of modern medicine. - World Health Organization,

  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. MRSA Cases by Year Number of new antimicrobial agents approved by the FDA for humans 18 16 16 14 Cases in Thousands 14 12 12 10 7 8 6 4 4 2 2 0 Adapted from: Data collected from hospital intensive care units that participate in the Infectious Diseases Society of America Clin Infect Dis. 2011; 52:S397-S428 National Nosocomial Infections Surveillance System of the Centers for Disease Control.

  6. MRSA resistance in a nutshell Cell Wall Chromosome PBP Penicillin Staphylococcus aureus

  7. MRSA resistance in a nutshell Cell Wall mecA gene Chromosome PBP2A Penicillin Methicillin Resistant Staphylococcus aureus

  8. Transcription mecA mRNA Translation PBP2 A Transcription MRSA mecA mRNA Translation PBP2 A Transcription STAPHYLOCIDE mecA mRNA Translation PBP2 A

  9. IMPROVING THE REGISTRY

  10. Staphylococcal Parts Selection Markers Promoters 1. ermM – Erythromycin resistance 1. sarA P1 – Strong constitutive 2. aadD – Kanamycin resistance 2. Xylose inducible promoter construct 3. spC – Spectinomycin resistance Origin of Replication Ribosome Binding Sites 1. pSK41 1. sodA RBS - S. aureus 2. Optimized TIR RBS - Theta Replictation - Low copy Terminators 1. sarA rho-independent Reporters - DsRed - YFP 9

  11. Staphylococcal Strain 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 10

  12. Reporter Gene: DsRed E. coli S. epidermidis -ve control 11

  13. E. coli-Staphylococcus Shuttle Vector Improved pSB1C3 by making it more versatile: pSB1C3 parts Parts we introduced S P RFP Expression Cassette (BBa_J04450) VR VF2 BBa_K1323017 Cm R oriV E. coli Erm R oriV S.aureus 12

  14. Shuttle Vector: Antibiotic Resistance • Stably maintained in S. epidermidis • Confers erythromycin resistance 13

  15. SILENCE DELIVER TRANSLATE

  16. SILENCE Transcription Translation Design

  17. Silence Transcription YFP mRNA Translation YFP 16

  18. Silence: CRISPRi dCas9-sgRNA complex blocks RNA polymerase 17

  19. Silence: CRISPRi Network 18

  20. Silence: CRISPRi Results 19

  21. Silence: CRISPRi Sensitivity • Sensitive to mRNA degradation rate Therefore… • Targeting of translation will improve silencing! 20

  22. Silence: RNAi sRNA-Hfq complex blocks ribosome 21

  23. Silence: RNAi Network 22

  24. Silence: RNAi Results 23

  25. Silence: Design CRISPRi Transcription YFP mRNA dCas9-sgRNA Translation Complex YFP 24

  26. Silence: CRISPRi P const xylose XylR sgRNA TT dCas9 TT pSB1A3 Amp R Erm R oriV E.coli oriV S. aureus 25

  27. Silence: Design CRISPRi Transcription YFP mRNA dCas9-sgRNA Translation RNAi Complex YFP Hfq-sRNA Complex 26

  28. Silence: RNAi P const xylose sRNA TT xylR Hfq TT pSB1A3 Amp R Erm R oriV E.coli oriV S. aureus 27

  29. Silence: RNAi Design YFP mRNA 3’ RBS Scar 5’ YFP CDS … sRNA 1 sRNA 2 sRNA 3 28

  30. Silence: RNAi Preliminary Tests pSB1A3 pSB3K3 Co- Transform E. coli DH5α Measure fluorescence 29

  31. Silence: RNAi Preliminary Test RFU/OD 600 YFP Alone Control sRNA1 sRNA2 sRNA3 30

  32. Silence: Future Directions • Characterize silencing systems in S. epidermidis • Integrate yfp into S. epidermidis genome • Incorporate the mecA gene regulation 31

  33. DELIVER Lab Design Modeling

  34. Conjugation in Staphylococcus Solid Surface Donor Recipient 33

  35. Deliver: Conjugation Advantages : Disadvantage : • Large carrying capacity • Not efficient • Independently propagates • Opportunity to contribute to an underdeveloped area of research 34

  36. Conjugation Parts: pGO1 pGO1: S. aureus conjugational plasmid oriT-nes : BBa_K1323003 RBS nes oriT TT 2.2 kb trs Region: Still in progress trs: 13.5 kb 35

  37. Conjugation Test Construct oriV S. aureus P S Erm R trs genes RBS nes RBS DsRed oriT TT TT pSBS1A3 oriV E.coli Amp R Filter Mating Assays Transconjugants Donor Recipients 36

  38. Deliver: Modeling Challenge: Modeling conjugation between cells spread across a lab plate or a patient’s skin 37

  39. 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 38

  40. Deliver: Agent Based Model Sufficient conjugation rate Staphylococcus conjugation rate Susceptible Staphylococcus MRSA t = 0 h t = 0 h 39

  41. Deliver: Agent Based Model Sufficient conjugation rate Staphylococcus conjugation rate Susceptible Staphylococcus MRSA t = 6 h t = 6 h 40

  42. Deliver: Agent Based Model Sufficient conjugation rate Staphylococcus conjugation rate Susceptible Staphylococcus MRSA t = 12 h t = 12 h 41

  43. Deliver: Agent Based Model Sufficient conjugation rate Staphylococcus conjugation rate Susceptible Staphylococcus MRSA t = 24 h t = 24 h 42

  44. Deliver: Agent Based Results 43

  45. Deliver: PDE Model Results 44

  46. Deliver: Future Uses of Model + Find igem-waterloo on GitHub! 45

  47. Deliver: Future Directions • Improve conjugation efficiency with error prone PCR mutagenesis and selective mating assays • Test conjugational efficiency in S. epidermidis 46

  48. TRANSLATE Market Viability Safety Adaptability

  49. Translate: Commercialization STAPHYLOCIDE Plasmid Conjugation Parts 48

  50. Translate: Commercialization 49

  51. Translate: Commercialization 50

  52. Translate: Commercialization β -Lactam Antibiotic 51

  53. Translate: Commercialization β -Lactam Antibiotic 52

  54. Translate: Adaptability 53

  55. SILENCE DELIVER TRANSLATE

  56. Accomplishments  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 55

  57. Accomplishments: Outreach Sir John A. Macdonald Secondary School Science Club High School Enrichment Program Lab Skills Video Series 56

  58. Acknowledgements Dr. Andrew Dr. Barbara Dr. Trevor Doxey Moffat Charles Dr. Marc Dr. Matthew Dr. Brian Aucoin Scott Ingalls 57

  59. Questions?

  60. References 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): 7429 – 3 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 O’Neill, A. J. (2009). Complete nucleotide sequence of pGO1, the prototype conjugative plasmid from the stap hylococci. 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-RNA – Guided 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. 59

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