Overview

• Goal
  • simulating emperical SIP data from validation experiments
    • SIP validations consisted of one or a few genomes
• Existing datasets
  • Lueders et al., 2004 (barkeri vs extorquens)
    • Lueders T, Manefield M, Friedrich MW. (2004). Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environmental Microbiology 6: 73–78.
  • Cupples et al., 2006 (E. coli & M. luteus)
    • http://www.sciencedirect.com/science/article/pii/S0944501306000176
  • Wawrik et al, 2009 (Synechococcus)
    • http://aem.asm.org/content/75/21/6662.long
  • Cupples et al., 2010 (Soil; Polaromonas)
    • http://aem.asm.org/content/76/3/956.full.pdf
  • Addison et al., 2010 (RNA; N. nitrogenifigens)
    • http://www.sciencedirect.com/science/article/pii/S0167701209003534
  • Andeer et. al., 2012 (Soil; E. coli, R rhodochrous; Microbacterium)
    • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255627/pdf/zam163.pdf

Study parameters

Lueders et al., 2004 (barkeri & extorquens)

• strain(s)
  • Methylobacterium extorquens AM1 DSM 1338
  • M. barkeri DSM 800
• isotope(s)
  • 13C
  • assumed 100% incorporation
• gradient
  • rotor
    • TV865 vertical rotor (Sorvall)
  • CsCl density
    • 1.725 g/ml
  • centrifugation
    • 20oC
    • >36h
    • 45 000 r.p.m. (177 000 gav)
  • fractionation volume
    • 400 ul
• Abundance quantification
  • DNA
  • primers
    • Ar109f/Ar915r (Lueders and Friedrich, 2003)
      • Ar109f (5′-ACK GCT CAG TAA CAC GT-3′)
      • Ar915r (5′-GTG CTC CCC CGC CAA TTC CT-3′)
    • Ba519f/Ba907 (Stubner, 2002)
      • Ba519f (CAGCMGCCGCGGTAANWC)
      • Ba907r (CCGTCAATTCMTTTRAGTT)

Cupples et al., 2006 (E. coli & M. luteus)

• strain(s)
  • E. coli (ATCC 15524)
• isotope(s)
  • 15N (E. coli & M. luteus)
  • 15N + 13C (E. coli)
  • assumed 100% incorporation

• gradient

  • rotor
    • VTi 65.2 vertical rotor (Beckman)
  • CsCl density
    • 1.725 g/ml ???
  • centrifugation
    • 20oC
    • 30 h
    • 228 000 gav
  • fractionation volume
    • 150 ul

• Abundance quantification

  • DNA
  • PicoGreen
    • shotgun fragment simulation

Wawrik et al, 2009 (Synechococcus)

• strain(s)
  • Synechococcus sp. strain WH7803
• isotope(s)
  • 15N
• gradient
  • rotor
    • Beckman VTI65.2
  • CsCl density
    • 1.701 g/ml
  • centrifugation
    • 140000 x g
    • 48 h
    • 20oC?
  • fractionation volume
    • 100 ul
• Abundance quantification
  • DNA
  • qPCR
    • fwd (CATCAAGCTGTCCGAG)
    • rev (TGTTGGCYGTGAAGCC)