SLIDE 1
Pathogen Detection Using an Engineered Contact-Dependent Inhibition System
John Errico 11/1/2014 iGEM
Human Practices
“I Nano Days”
SB County Science Fair
Using an Engineered Contact-Dependent Inhibition System John - - PowerPoint PPT Presentation
Using an Engineered Contact-Dependent Inhibition System John - - PowerPoint PPT Presentation
Pathogen Detection Using an Engineered Contact-Dependent Inhibition System John Errico 11/1/2014 iGEM Human Practices I Nano Days SB County Science Fair What is the Allosphere? Image from
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What is the Allosphere?
A simulated 3D immersive environment at UCSB
Image from http://www.nsf.gov/discoveries/disc_images.jsp?cntn_id=121535&org=NSF For more information: http://www.allosphere.ucsb.edu/
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CDI stands for contact-dependent inhibition
- A bacterial defense and communication strategy
- Injected
toxins act as non-specific DNases, RNases, etc.
- Mechanisms for self-strain protection against toxin (CdiI)
Ruhe, Low, & Hayes. Trends in Microbiology (2013)
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CDI is composed of 3 genes
- CdiB is a membrane-
bound ‘base’ for CdiA
- CdiI is an immunity protein specific
for upstream toxin that prevents self-killing
Conserved VENN Sequence CT - Toxin Sequence CdiI - Immunity Protein ‘stick’ ‘base’
Ruhe, Low, & Hayes. Trends in Microbiology (2013)
- CdiA is the ‘stick’ with toxin attached to end, cleaved
@ conserved VENN sequence – binds BamA
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CT may need a permissive factor to become active
When CT binds CysK (permissive factor), enzymatic activity is enabled Both CysK and Immunity can bind CT to form a ternary complex
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One half of Adenylate Cyclase (T25) fused to protein X, other half (T18) fused to Protein Y
Bacterial two-hybrid systems detect protein-protein interactions
If Proteins X/Y interact, Adenylate Cyclase forms cAMP production identifies protein-protein interactions
Adapted from Karimova G et al. PNAS 1998;95:5752-5756
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cAMP production turns on gene synthesis
Pap operon is a chromosomal promoter dependent on cAMP –
- nly source of cAMP is
from our system Gene of interest must be transduced using a bacteriophage
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CysK-T25 was made using OE-PCR
CysK (~800bp) T25 (~1000bp)
Overlapping Primers
CysK forward primer w/ EcoRI site
T25 reverse primer w/ PstI site
PCR, longer extension period Fused CysK-T25 product
Fused PCR product ligated into Tetracycline resistant backbone, transformed, checked with restriction analysis:
1.8Kbp = CysK-T25
Linker = Gly-Thr-Gly-Ser
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I-T18 was extracted from pre-existing plasmid with PCR
Ligation into Ampicillin resistant backbone, transformation, check with restriction analysis:
I
PstI
T18
U T25 5’ 3’ Standard PCR
I T18
EcoRI EcoRI PstI 5’ 3’
Faint 1Kbp = I-T18
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Bacteriophage steal DNA randomly from a host
Introduce GFP-grown phage to cell line, select for KanR colonies
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Bacteria did not glow upon contact – Why?
Linker length may be not be long enough - Ternary vs. Binary
Vs.
Endogenous CysK may be interfering – competitive antagonist Couldn’t control by transfecting commercial binary two- hybrid construct – Conflicting antibiotic resistances
Adapted from Karimova G et al. PNAS 1998;95:5752-5756
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The Future – a very customizable system
- Contact-dependent bacterial strain – Can include as
many immunities as necessary
- Secondary messenger produced –cGMP?
- Gene synthesized – multi-gene cascade systems?
- Antibiotic production? – extremely specific killing
Think T-cells
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Pathogen Detection Using an Engineered Contact-Dependent Inhibition System
John Errico 11/1/2014 iGEM
Gene Expression
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Acknowledgements
Advisors: David Low, Omar Saleh Mentors: Christina Beck, Dan Nguyen Lab space: Chris Hayes The Team: Zachary Haynes, Travis Smith, Hiro Sparks, Katie Lee, Sarah Lensch, Andrew Ballin, Daniel Reinhart, Colton Bracken, & Tsuyoshi Kohlgruber And of course… iGEM!
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