fluorescent detection of cadmium in water...
TRANSCRIPT
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Fluorescent
Detection of
Cadmium in
Water Supplies
• Gaston Day School
• 2013 iGEM Team
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Our Team• Advisor: Ms. Byford
• Seniors:
• Steven Allen (Team Co-Leader)
• Parth Patel (Team Co-Leader)
• Gordon Ellison
• Juniors:
• Will Reiber
• Jewel Wasson
• Paige Brown
• India Mackinson
• Elizabeth England
• Sophomores:• Claire Lamadrid
• Heather Cheung
• Associate Members:• Misbah Ahmad
• Nana Boateng
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Project Choice
• BioSafety Level 1
• High School Classroom Environment
• Parts available in the registry
• Local Funding
• Relevant to our area
• Agriculture
• Industry
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Original Intentions
• Two teams
• Multiple Projects
• Alternative Energy
• Heavy Metal Detection
• Time and resources
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Original Intentions
• Biofuels
• AceE, AceF, lpd genes
• 1-butanol
• E. Coli genes BioBricks
• Primers
• Assemble one large operon
• Overexpression?
• Efficiency?
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
ATSDR List
• Agency for Toxic Substances and Disease
Registry
• Division of CDC
Substance Priority List
1. Arsenic 6. Benzene
2. Lead 7. Cadmium
3. Mercury 8. Benzo (a) pyrene
4. Vinyl Chloride 9. Polycyclic Aromatic
Hydrocarbons
5. Polychlorinated Biphenyls 10. Benzo (b) Fluoranthene
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Cadmium
• Sources
• Industry
• Mining / Smelting
• Zinc, Lead, Copper
• Water System
• Soil / Rock Erosion
• Nickel-Cadmium Batteries
• Metal-Coatings
• Federal Limit = 0.005 mg/L
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Cadmium
• Health Effects:
• Affected Systems:
• Cardiovascular
• Respiratory
• Gastrointestinal
• Known Carcinogen
• Can cause cancer in any affected system
• Stronger effects in children
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Safety
• Is our number one concern!
• High school environment
• High traffic
• Protective Measures
• Safety Training
• Standard Safety Procedures
• Sterile Technique
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Cadmium Sensor• Mutagenic PCR
• U.C. Davis 2010 Protocol
• Random Mutations through varied
concentrations, buffers, and polymerase
specificity
• Possibility of increased sensitivity in
promoter or reduced function
• No improvement in sensitivity
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Cadmium Sensor
• Cambridge 2007 Sensitivity Tuners
• Bacteriophage Activators and Promoters
• Should not affect cell activity
• 2 of 15 combinations chosen
• Ogr and Delta Activators combined with
PO Promoter
• Hoped for 30 – 40 fold sensitivity
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Construction
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Transcribe
Here
Sensor Model
Cd
Transcription
FactorsRibosome
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Testing Protocol • 4 mL LB Culture in Plastic Tubes
• Positive Control
• Last year’s detector
• Negative Control
• DH5a
• Concentrations
• CADMIUM CHLORIDE?? 1m???
• 1 mM
• 5 mM
• 10 mM
• 50mM
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Complication One • Control (left) & 50 mM Cadmium Chloride (right)
• LB Fluoresces Naturally!
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Results
• Luria Broth Fluoresces naturally
• Account for growth & GFP
• Protocol
• Spin cells down for 5 minutes
• Resuspend in 1 mL PBS (1X)
• Measure in Fluorescence per OD 600
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Results
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Our Results
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Interpreting Our Results
• Initial construct is not more sensitive despite
promising initial results
• Less growth at 5 mM but increased at 50
mM
• Why did this happen?
• Possible solubility issue at 50 mM
• Cambridge also saw reduced growth
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Complications, Complications,
Complications
• Detector is not functioning properly
• Test results
• Fungus over the summer
• Complete decontamination
• New solutions, antibiotics, cultures, etc.
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Now What?
• Completely started over
• Transform necessary parts
• Repeat 3X
• Digest
• Size check
• Ligation
• Transformation
• Digest
• PCR
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Our BioBrick
• BBa_K1042013
• PO Promoter + GFP with Stops
• Picture of PCR / Digest with Bands!
• Accurate Size
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Our New Parts
• Already submitted downstream part
• K1042013
• Upstream part
• Cadmium Promoter + P2 Ogr activator
• Entire construct
• K1042015
• Cadmium Responsive GFP + Ogr activator
• Not yet tested
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Bands!
• K1042015
• 1413 base pairs
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Solutions & Potential Use• Ligate Cadmium Promoter + ogr activator with
Bba_K1042013• Full detector
• Appropriate antibiotic resistances
• Full detector• Easy to interpret results
• Self-contained kit…
• Marketing
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Introduction Cadmium
Background
Our Project Results &
Complications
Solutions & the
Future
Future Plans
• Sensitivity Tuners?
• Cell suicide gene
• Biofuels
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Sponsors
Sandra and Bill Hall
New England Biolabs
SignSmart
Gaston Day School