DNA Stable-Isotope Probing
Dr. Josh D. Neufeld Department of Biology University of Waterloo June 17, 2010 Metagenomics workshop Canadian Society for Microbiologists McMaster University, Hamilton, Ontario
DNA Stable-Isotope Probing
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How SIP works
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Advice for key steps: Incubation, Separation, Analysis
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What I wish I had known earlier
1 • 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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How SIP works
Incubate sample Fractionate DNA Characterize DNA
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Advice for key steps
Soil and sediment 5–500 mmol 13C per g
Aquatic samples 1–100 mmol 13C per liter
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)
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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).
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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Advice for key steps
Neufeld et al. 2008 Appl. Environ. Microbiol. 13C
2-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.
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Advice for key steps
el Zahar Haichar et al. 2007 Environmental Microbiology 13C -cellulose n
produced from 13C6-glucose by Gluconacetobacter xylinus.
Labelled cells, DNA, proteins, lipids, pollutants
Ethidium bromide and UV exposure
Ethidium bromide and UV exposure
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Advice for key steps
1 13CH
3OH
0.1
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1 12CH
0.1
3OH
Low µM concentrations used Estimated seawater concentration: 0.1-0.3 µM
heavy
light
heavy
light
13 MOH
12 MOH
1μM 13MOH
0.1μM 12 MOH
13 MOH
12 MOH
8 10 8 10 8 10 8 10 8 10 8 10
Ladder
Ladder
10μM
12C heavy
2 weeks
1 month
2 months
6-glucose light
13C -glucose 6 heavy
light
12C
n-cellulose
heavy
2 weeks
1 month
2 months
light
13C
n-cellulose
heavy
light
Glycogen Full recovery Visible pellet
Neufeld JD et al. Environ. Microbiol. In press.
Multiple displacement amplification (MDA)
Multiple displacement amplification (MDA) = chimeras
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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
What I wish I had known earlier
3 L
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12
L
12,000 bp 5,000 bp 2,000 bp
13C
M. capsulatus
12C
S. meliloti
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
[email protected] 