STAPHYLOCIDE : Delivering Antibiotic Resistance Gene Silencing - - PowerPoint PPT Presentation

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STAPHYLOCIDE : Delivering Antibiotic Resistance Gene Silencing - - PowerPoint PPT Presentation

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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,

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STAPHYLOCIDE:

Delivering Antibiotic Resistance Gene Silencing Mechanisms to a MRSA Population using Bacterial Conjugation

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"The problem is so serious that it threatens the achievements of modern medicine. ”

  • World Health Organization, Antimicrobial Resistance:

Global Report on Surveillance 2014

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80 461

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11 285

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SLIDE 5 Infectious Diseases Society of America Clin Infect Dis. 2011; 52:S397-S428 Adapted 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

2 4 6 8 10 12 14 16 18

Number of new antimicrobial agents approved by the FDA for humans Cases in Thousands

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MRSA resistance in a nutshell

Penicillin PBP Chromosome Cell Wall

Staphylococcus aureus

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MRSA resistance in a nutshell

Methicillin Resistant Staphylococcus aureus

mecA gene Penicillin PBP2A Chromosome Cell Wall

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SLIDE 8 mecA mRNA Transcription Translation PBP2 A mecA mRNA Transcription Translation PBP2 A mecA mRNA Transcription Translation PBP2 A

MRSA STAPHYLOCIDE

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IMPROVING THE REGISTRY

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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
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  • 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

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Reporter Gene: DsRed

  • E. coli
  • S. epidermidis
  • ve control
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  • E. coli-Staphylococcus Shuttle Vector

BBa_K1323017

ErmR

  • riVE. coli

CmR

  • riVS.aureus

P S RFP Expression Cassette (BBa_J04450) VF2 VR

Improved pSB1C3 by making it more versatile:

pSB1C3 parts Parts we introduced

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Shuttle Vector: Antibiotic Resistance

  • Stably maintained in
  • S. epidermidis
  • Confers erythromycin

resistance

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DELIVER SILENCE TRANSLATE

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SILENCE

Design Translation Transcription

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Silence

YFP mRNA Transcription Translation YFP

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Silence: CRISPRi

dCas9-sgRNA complex blocks RNA polymerase

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Silence: CRISPRi Network

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Silence: CRISPRi Results

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Silence: CRISPRi Sensitivity

  • Sensitive to mRNA

degradation rate Therefore…

  • Targeting of

translation will improve silencing!

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Silence: RNAi

sRNA-Hfq complex blocks ribosome

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Silence: RNAi Network

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Silence: RNAi Results

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Silence: Design

YFP mRNA Transcription Translation YFP

CRISPRi

dCas9-sgRNA Complex

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pSB1A3

AmpR

  • riVE.coli

ErmR

  • riVS. aureus
TT

sgRNA

Pconst

Silence: CRISPRi

dCas9 xylose XylR

TT
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Silence: Design

YFP mRNA Transcription Translation YFP

RNAi CRISPRi

dCas9-sgRNA Complex Hfq-sRNA Complex

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pSB1A3

AmpR

  • riVE.coli

ErmR

  • riVS. aureus
TT

sRNA

Pconst

Silence: RNAi

Hfq xylose

TT

xylR

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Silence: RNAi Design

YFP CDS Scar RBS YFP mRNA 5’ 3’ …

sRNA 1 sRNA 2 sRNA 3

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Co- Transform

Silence: RNAi Preliminary Tests

pSB3K3

  • E. coli DH5α

Measure fluorescence

pSB1A3

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Silence: RNAi Preliminary Test

YFP Alone Control sRNA1 sRNA2 sRNA3 RFU/OD600

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  • Characterize silencing systems in S. epidermidis
  • Integrate yfp into S. epidermidis genome
  • Incorporate the mecA gene

regulation

Silence: Future Directions

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DELIVER

Modeling Lab Design

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Conjugation in Staphylococcus

Solid Surface

Recipient Donor

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Deliver: Conjugation

Advantages:

  • Large carrying capacity
  • Independently propagates
  • Opportunity to contribute

to an underdeveloped area of research Disadvantage:

  • Not efficient
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Conjugation Parts: pGO1

pGO1: S. aureus conjugational plasmid

  • riT-nes: BBa_K1323003
  • riT
nes RBS TT

2.2 kb

trs Region: Still in progress

trs: 13.5 kb

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Conjugation Test Construct

pSBS1A3

ErmR

AmpR

  • riVE.coli

P

DsRed TT RBS
  • riVS. aureus
nes TT RBS
  • riT
trs genes

S Recipients

Donor Transconjugants Filter Mating Assays

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Deliver: Modeling

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

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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

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Deliver: Agent Based Model

Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 0 h t = 0 h

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Deliver: Agent Based Model

Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 6 h t = 6 h

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Deliver: Agent Based Model

Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 12 h t = 12 h

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Deliver: Agent Based Model

Staphylococcus conjugation rate Susceptible Staphylococcus MRSA Sufficient conjugation rate t = 24 h t = 24 h

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Deliver: Agent Based Results

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Deliver: PDE Model Results

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Deliver: Future Uses of Model

+

Find igem-waterloo on GitHub!

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  • Improve conjugation efficiency with error prone

PCR mutagenesis and selective mating assays

Deliver: Future Directions

  • Test conjugational efficiency in
  • S. epidermidis
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TRANSLATE

Adaptability Safety Market Viability

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Translate: Commercialization

STAPHYLOCIDE Plasmid

Conjugation Parts

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Translate: Commercialization

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Translate: Commercialization

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Translate: Commercialization

β-Lactam Antibiotic

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Translate: Commercialization

β-Lactam Antibiotic

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Translate: Adaptability

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DELIVER SILENCE TRANSLATE

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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

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Accomplishments: Outreach

High School Enrichment Program

Science Club

Lab Skills Video Series

Sir John A. Macdonald Secondary School

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Acknowledgements

  • Dr. Marc

Aucoin

  • Dr. Brian

Ingalls

  • Dr. Matthew

Scott

  • Dr. Trevor

Charles

  • Dr. Barbara

Moffat

  • Dr. Andrew

Doxey

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Questions?

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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 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
  • n 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.

References

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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): 1173–83.
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