the problem is so serious that it threatens the...

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

16

Silence

YFP mRNA

Transcription

Translation

YFP

17

Silence: CRISPRi

dCas9-sgRNA complex blocks RNA polymerase

18

Silence: CRISPRi Network

19

Silence: CRISPRi Results

20

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

31

• Characterize silencing systems in S. epidermidis

• Integrate yfp into S. epidermidis genome

• Incorporate the mecA gene regulation

Silence: Future Directions

DELIVERModelingLab Design

33

Conjugation in Staphylococcus

Solid Surface

RecipientDonor

34

Deliver: Conjugation

Advantages:• Large carrying capacity • Independently propagates• Opportunity to contribute

to an underdeveloped area of research

Disadvantage:• Not efficient

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

36

Conjugation Test Construct

pSBS1A3

ErmR

AmpRoriVE.coli

P DsRed TTRBS oriVS. aureusnes TTRBSoriTtrs genes S

RecipientsDonor Transconjugants

Filter MatingAssays

37

Deliver: Modeling

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

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

39

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

43

Deliver: Agent Based Results

44

Deliver: PDE Model Results

45

Deliver: Future Uses of Model

+

Find igem-waterloo on GitHub!

46

• Improve conjugation efficiency with error prone PCR mutagenesis and selective matingassays

Deliver: Future Directions

• Test conjugational efficiency in S. epidermidis

TRANSLATE

AdaptabilitySafetyMarket Viability

48

Translate: Commercialization

STAPHYLOCIDE Plasmid

Conjugation Parts

49

Translate: Commercialization

50

Translate: Commercialization

51

Translate: Commercialization

β-LactamAntibiotic

52

Translate: Commercialization

β-LactamAntibiotic

53

Translate: Adaptability

DELIVERSILENCE TRANSLATE

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

56

Accomplishments: Outreach

High School Enrichment ProgramScience Club Lab Skills Video Series

Sir John A. MacdonaldSecondary School

57

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

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,

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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