the problem is so serious that it threatens the...
TRANSCRIPT
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, Antimicrobial Resistance: Global Report on Surveillance 2014
80 461
11 285
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
1614
12
7
42
0
2
4
6
8
10
12
14
16
18
Number of new antimicrobial agents approved by the FDA
for humans
Cas
es
in T
ho
usa
nd
s
MRSA resistance in a nutshell
PenicillinPBP
Chromosome
Cell Wall
Staphylococcus aureus
MRSA resistance in a nutshell
Methicillin Resistant Staphylococcus aureus
mecA gene
PenicillinPBP2A
Chromosome
Cell Wall
mecA mRNA
Transcription
Translation
PBP2
A
mecA mRNA
Transcription
Translation
PBP2
A
mecA mRNA
Transcription
Translation
PBP2
A
MRSA
STAPHYLOCIDE
IMPROVING THE REGISTRY
9
Promoters1. sarA P1 – Strong constitutive2. Xylose inducible promoter construct
Ribosome Binding Sites1. sodA RBS2. Optimized TIR RBS
Terminators1. sarA rho-independent
Staphylococcal Parts
Selection Markers1. ermM – Erythromycin resistance2. aadD – Kanamycin resistance3. spC – Spectinomycin resistance
Origin of Replication1. pSK41
- S. aureus- Theta Replictation- Low copy
Reporters- DsRed- YFP
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
11
Reporter Gene: DsRed
E. coli S. epidermidis -ve control
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
13
Shuttle Vector: Antibiotic Resistance
• Stably maintained in
S. epidermidis
• Confers erythromycin
resistance
DELIVERSILENCE TRANSLATE
SILENCE
DesignTranslationTranscription
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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!
21
Silence: RNAi
sRNA-Hfq complex blocks ribosome
22
Silence: RNAi Network
23
Silence: RNAi Results
24
Silence: Design
YFP mRNA
Transcription
Translation
YFP
CRISPRi
dCas9-sgRNA
Complex
25
pSB1A3
AmpRoriVE.coli ErmR oriVS. aureus
TTsgRNA
Pconst
Silence: CRISPRi
dCas9
xylose
XylR TT
26
Silence: Design
YFP mRNA
Transcription
Translation
YFP
RNAi
CRISPRi
dCas9-sgRNA
Complex
Hfq-sRNA
Complex
27
pSB1A3
AmpRoriVE.coli ErmR oriVS. aureus
TTsRNA
Pconst
Silence: RNAi
Hfq
xylose
TTxylR
28
Silence: RNAi Design
YFP CDSScarRBSYFP mRNA
5’ 3’
…
sRNA 1
sRNA 2
sRNA 3
29
Co- Transform
Silence: RNAi Preliminary Tests
pSB3K3
E. coli DH5αMeasure fluorescence
pSB1A3
30
Silence: RNAi Preliminary Test
YFP Alone Control sRNA1 sRNA2 sRNA3
RF
U/O
D600
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• Characterize silencing systems in S. epidermidis
• Integrate yfp into S. epidermidis genome
• Incorporate the mecA gene regulation
Silence: Future Directions
DELIVERModelingLab Design
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Conjugation in Staphylococcus
Solid Surface
RecipientDonor
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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
oriT-nes: BBa_K1323003
oriT nesRBS TT
2.2 kb
trs Region: Still in progress
trs: 13.5 kb
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Conjugation Test Construct
pSBS1A3
ErmR
AmpRoriVE.coli
P DsRed TTRBS oriVS. aureusnes TTRBSoriTtrs genes S
RecipientsDonor Transconjugants
Filter MatingAssays
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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 ModelStaphylococcus conjugation rate
Susceptible Staphylococcus
MRSA
Sufficient conjugation rate
t = 0 ht = 0 h
40
Deliver: Agent Based ModelStaphylococcus conjugation rate
Susceptible Staphylococcus
MRSA
Sufficient conjugation rate
t = 6 h t = 6 h
41
Deliver: Agent Based ModelStaphylococcus conjugation rate
Susceptible Staphylococcus
MRSA
Sufficient conjugation rate
t = 12 h t = 12 h
42
Deliver: Agent Based ModelStaphylococcus 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 matingassays
Deliver: Future Directions
• Test conjugational efficiency in S. epidermidis
TRANSLATE
AdaptabilitySafetyMarket 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-LactamAntibiotic
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Translate: Commercialization
β-LactamAntibiotic
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Translate: Adaptability
DELIVERSILENCE 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 ProgramScience Club Lab Skills Video Series
Sir John A. MacdonaldSecondary School
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Acknowledgements
Dr. Marc Aucoin
Dr. Brian Ingalls
Dr. Matthew Scott
Dr. Trevor Charles
Dr. Barbara Moffat
Dr. Andrew Doxey
Questions?
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): 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
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
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References
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