www.sciencemag/cgi/content/full/330/6009/1413/DC1
Supporting Online Material for
Direct Exchange of Electrons Within Aggregates of an Evolved Syntrophic
Coculture of Anaerobic Bacteria
Zarath M. Summers, Heather Fogarty, Ching Leang, Ashley E. Franks, Nikhil S. Malvankar,
Derek R. Lovley*
*To whom correspondence should be addresd. E-mail: dlovley@microbio.umass.edu
北京大学毕业典礼
Published 3 December 2010, Science330, 1413 (2010)
心理自测DOI: 10.1126/science.1196526
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This PDF file includes:
Materials and Methods
Figs. S1 to S6
References
Fluorescent in situ hybridization (FISH). Cell aggregates were immerd in fixative (2% paraformaldehyde and 0.5% glutaraldehyde in 50 mM PIPES at pH 7.2) for one hour at room temperature, and were then dehydrated in a graded ethanol ries (30, 50, 70, 80, 100% ethanol, 30 min each stage with gentle agitation). The dehydrated samples were infiltrated and embedded in BMM (butyl-methyl-methacrylate).Semi-thin ctions (500 nm) of BMM embedded samples were mounted on glass slides and gently dried at 37 C°. BMM was partially dissolved in acetone for 30 conds before hybridization onto microscope slides. The thin ctions were hybridized as previously described (S11), with the following probes: GEO1: 5’-[cy3]AGAATCCAAGGACTCCGT-3’, specific for G. metallireducens, and GEO2: 5’-[cy5]GAAGACAGGAGGCCCGAAA-3’, specific for G. sulfurreducens.Samples were imaged with a Leica TCS SP5 microscope
(Leica Microsystems GmbH, Wetzlar, Germany) with HCX PL APO CS 20 (numerical aperture 0.7), HCX APO 63 (numerical aperture: 0.9) and HCX PL APO CS 100 x (numerical aperture 1.4) objectives. Concutive line scanning was ud to detect Cy3 (excitation 543 nm/emission 559-622 nm) and Cy5 (excitation 633 nm/emission 650-750) fluorescence. All images were procesd and analyzed with Leica LAS AF software (Leica).
Immunogold labeling and transmission electron microscopy. OmcS was localized as previously desc
ribed (S12). Briefly, aggregates fixed and dehydrated as described above were embedded in LR White, thin ctioned (60-80 nm), and ctions mounted on colloidion coated gold gilded copper grids, and immunogold labeled with anti-OmcS rabbit polyclonal antibodies and then with anti-rabbit IgG conjugated with 10-nm gold condary antibody. Cells were stained with uranyl acetate. Preparations were obrved with a JEOL 100S transmission electron microscope at 80 kV.
Quantitative PCR analysis. Genomic DNA was extracted using the MasterPure Complete DNA Purification kit (Epicentre Biotechnologies) according to the manufacturer’s directions. Primers unique to each species were ud to determine the proportions of each within the aggregate samples: Gmet_F 5’-ATGGCCCACATCTTCATCTC-3’, Gmet_R 5’-TGCATGTTTTCATCCACGAT-3’, Gsulf_F 5’-CCAGCTACGCCTACTTCTTCTTT-3’, Gsulf_R 5’-AAGCTGTGGTTCAGGAGGTATTT-3’. Primers that amplified from both species were ud to determine total combined species prent: Geo16S_F 5’-GAGGTACCGTCAAGACCAA-3’, Geo16S_R 5’-GCCACACTGGAACTGAGACA-3’. QPCR was performed as previously described (S13) on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems).
Sequencing of Evolved Co-Culture Whole-genome quencing was performed using Illumina Sequencing as previously described (S14). One of nine replicates was quenced and compared to
如何挑选玉镯the quences of the two ancestral WT strains. The other eight replicates were then screened for the point mutation found during quencing with Sanger quencing.
Cytochrome Heme Stain Analysis and Western Blot. Whole-cell lysates (5µg) were parated by 12.5% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). Heme staining was carried out as previously described (S15, S16) using N, N, N’, N’-tetramethylbenzidine. A replicate of the SDS-PAGE was immunoblotted, and probed with OmcS-specific antirum (S12) using the One-Step Western Kit (GeneScript Co.) according to the manufacturer’s directions. An additional replicate of the 12.5% SDS-PAGE was stained with Coomassie blue.酒美
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Conductance Measurements. The conductance of aggregates was measured with the same method previously described for measuring the conductance of gold nanowires
告别信奉行是什么意思(S15) in which the material of interest is placed on two gold electrodes parated by a 50 µm non-conductive gap. Aggregates were placed on the electrode gap with a micropipette and the residual buffer was removed with filter paper. The aggregates formed a smooth continuous film on the electrodes. Voltage was applied across the gap using Keithley 2400 source meter. Voltage was scanned from -0.5V to +0.5V in steps of 0.05 V, then from +0.5V to -0.5V. For each measurement, c
urrent was measured 10 conds after tting the voltage to allow for the exponential decay of the transient ionic current in the gap and to measure steady-state electronic current (S18). Igor Pro (Wavemetrics Inc. OR USA) was ud for data fitting and analysis. Aggregate attachment to gold electrodes was documented using confocal microscopy. Aggregates were stained with syto9 from LIVE/DEAD BacLight bacterial viability kit Molecular Probes (Eugene, OR, USA) as previously described (S19). Aggregates were imaged with a Leica TCS SP5 microscope (Leica Microsystems GmbH,Wetzlar, Germany) and images where procesd and analyzed with Leica LAS AF software (Leica). Concutive line scanning was ud to detect syto 9 (excitation 488nm/ excitation 520 nm) and reflection from the gold electrode surface (561 nm).
sulfurreducens (red) in the co-cultures at different stages of transfer.
