dna stable-isotope probing
TRANSCRIPT
June 17, 2010
Metagenomics workshop Canadian Society for Microbiologists
McMaster University, Hamilton, Ontario
Dr. Josh D. Neufeld Department of Biology University of Waterloo
DNA Stable-Isotope Probing
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1 How SIP works
Advice for key steps: Incubation, Separation,
Analysis
What I wish I had known earlier
DNA Stable-Isotope Probing
• Links phylogeny with function – Who eats what?
• No requirement for cultivation • Applied to diverse environments
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Dunford and Neufeld, 2010 (In press)
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1 How SIP works
Incubate sample
Fractionate DNA
Characterize DNA
Soil and sediment 5–500 mmol 13C per g
Aquatic samples 1–100 mmol 13C per liter
2 Advice for key steps
Acetate in Lake Kinneret (Schwarz et al., 2007)
Acetate Florida Everglades (Chauhan and Ogram, 2006)
Cellulose in agricultural soil (el Zahar Haichar et al., 2007)
Benzene in contaminated groundwater (Kasai et al., 2006)
Organic substrates in marine sediments (Webster et al., 2006)
Atmospheric methane in an upland soil (Maxfield et al., 2006)
2,4-dichlorophenoxyacetic acid in a field soil (Cupples and Sims, 2007)
Pyrene in a soil bioreactor (Singleton et al., 2006)
Bicarbonate in an estuarine sediment (Freitag et al., 2006)
Propionate in a wastewater bioreactor (Meyer et al., 2006)
Acetate/methanol in activated sludge (Osaka et al., 2006)
Wheat residue in soil (Bernard et al., 2007)
CO2 to plants and the rhizosphere (Cadisch et al., 2005; Lu and Conrad, 2005; Rangel-Castro et al., 2005).
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Carbon dioxide $100 per L
Carbon monooxide $275 per L
Glucose $50-$100 per g
Fructose $500 per g
Arabinose $1200 per g
Xylose $800 per g
Cellobiose $5000 per g
Sigma Aldrich Cambridge Isotope Laboratories
Omicron Biochemicals Inc
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Neufeld et al. 2008 Appl. Environ. Microbiol.
2 Advice for key steps
13C2-dimethylsulfide was prepared by a method adapted from that described for labeled
dimethyl sulfoxide synthesis. Sodium sulfide
nonahydrate and 5 g of 13C1-methyl iodide.
Flame ionization detector.
el Zahar Haichar et al. 2007 Environmental Microbiology
2 Advice for key steps
13Cn-cellulose produced from 13C6-glucose by Gluconacetobacter xylinus.
Labelled cells, DNA, proteins, lipids, pollutants
Ethidium bromide and UV exposure
Ethidium bromide and UV exposure
Ethidium bromide and UV exposure
Ethidium bromide and UV exposure
10 1 0.1
13CH3OH
10 1 0.1
12CH3OH
Low µM concentrations used Estimated seawater concentration: 0.1-0.3 µM
2 Advice for key steps
heavy light heavy light
Lad
der
Lad
der
8 10 8 10 8 10 8 10 8 10 8 10
10 μ M
13 MOH 12 MOH
1 μ M
13 MOH 12 MOH
0.1 μ M
13 MOH 12 MOH
2 weeks
1 month
2 months
12C6-glucose 13C6-glucose heavy light heavy light
12Cn-cellulose 13Cn-cellulose heavy light heavy light
2 weeks
1 month
2 months
Glycogen
Full recovery
Visible pellet
Neufeld JD et al. Environ. Microbiol. In press.
Multiple displacement amplification (MDA)
Multiple displacement amplification (MDA) = chimeras
3 What I wish I had known earlier
What to buy?
Interpretation/Contamination
Vertical rotor Vti 65.2 (Beckman Coulter)
Reichart AR200 Digital Refractometer
VWR; ~$1000
Braintree Scientific: $800 AnytimeLab Traders: $250
87 6532 1 9 10 11 12 4L
12C S. meliloti
13C M. capsulatus
12,000 bp 5,000 bp
2,000 bp
L
3 What I wish I had known earlier
Denitrifica*on SIP in agricultural soils Biol 499 project , U of G collabora*on
Link who to what Metagenomics
(chimeras) Equipment
Interpretation
Acknowledgements
Eric Dunford (Waterloo) Hataichanok Sae Yang (Waterloo)
Colin Murrell (Warwick) Marc Dumont (Warwick)
Yin Chen (Warwick)
CFI NSERC Strategic Projects