Gf a filter

[³H]-α-bungarotoxin ([³H]-αBTX; 56 Ci/mmol; Tocris Bioscience, Bristol, UK) was a gift from Syngenta (Bracknell, UK). Radioligand binding to transiently transfected tsA201 cells was performed essentially as described previously (Lansdell and Millar, 2004 (link)). Transfected cells were resuspended in Hank's buffered saline solution (Gibco, Paisley, UK) containing 1% BSA and incubated with [³H]-αBTX for 2 h at room temperature in a total volume of 300 μl. Non-specific binding was determined in the presence of nicotine (1 mM) and carbamylcholine (1 mM). Saturation binding experiments were performed by incubating triplicate samples of transfected cells with varying concentrations of [³H]-αBTX. Radioligand binding was assayed by filtration onto Whatman GF/A filters (pre-soaked in 0.5% polyethylenimine), followed by rapid washing with phosphate-buffered saline (Oxoid, Basingstoke, UK) using a Brandel cell harvester. Bound radioligand was quantified by scintillation counting. Curves for equilibrium binding were fitted using GraphPad Prism (GraphPad Software, San Diego, USA).

Activation of the S-phase checkpoint was determined using the method of Garg et al [14 ]. HeLa cells were prelabeled for ~24 hr by culture in DMEM containing 10 nCi/mL [¹⁴C]-thymidine. Following prelabeling, the medium containing [¹⁴C]-thymidine was replaced with normal DMEM, and the cells were incubated for 6 hr. The cells were then treated with Cr for 4 hours, followed by pulse-labeling with 2.5 μCi/mL [³H]-thymidine for 15 min. Following pulse-labeling, cells were harvested, washed twice with PBS, and fixed in cold (-4 °C) 70% methanol. Afterwards, the cells were applied to Whatman GF/A filters which were then rinsed sequentially in 70% and 95% methanol, air dried, and then placed in glass vials containing 3 ml of scintillation fluid. The filters were then analyzed on a Beckman scintillation counter with windows set to record both ¹⁴C and ³H dpm. The measure of DNA synthesis was derived from resulting ratios of³H cpm to ¹⁴C cpm and corrected for counts resulting from channel crossover.

A total of 10 L of water was pre-filtered through ∼1.6 mm GF/A filters (Whatman) and cells were collected on 0.22 μm Sterivex filters (Millipore) using a peristaltic pump. Sterivex filters were stored at −80°C until DNA extraction. DNA was extracted as described in [29] (link). Briefly, filters were treated with lysis buffer (50 mM Tris-HCl, 40 mM EDTA, and 0.75 M sucrose) and incubated with 1 mg/ml lysozyme at 37°C for 30 min. Samples were subsequently incubated with 1% SDS, 10 mg/ml proteinase K, and 150 mg/ml RNAse for 2 h rotating at 55°C. DNA was extracted from the lysate with phenol and chloroform, precipitated with ethanol and eluted in TE buffer. DNA yield was about 1.5 μg per liter of water filtered. For the metagenome, ∼5 μg of the total DNA aliquot was sequenced using the Illumina GA-II sequencers at the Emory University Genomics Facility, providing paired-end reads with an average length of 100 bp (Table S4 in File S1). 16S rRNA gene amplicon pyrosequencing (see below) was run on the GS-FLX 454 Titanium platform, also at the Emory University Genomics Facility.

The DCBMs were produced as HFBI-DCBM fusions in T. reesei as described in details in earlier work of the group19 (DCBM sequences in Supplementary Figure S1) and the following transformants were used: VTT-D-133335 (HFBI-DCBM-12), VTT-D-133336 (HFBI-DCBM-24), and VTT-D-133337 (HFBI-DCBM-48). The strains were then cultivated in 7 L bioreactors on media containing 50 vol-% spent grain extract, 60 g/L lactose, 1 g/L yeast extract, 4 g/L KH2PO4, 2.8 g/L (NH4)2SO4, 0.6 g/L MgSO4 ∙ 7H2O, 0.8 g/L CaCl2 ∙ 2H2O, 2 ml/L trace solution. The pH was let to drop from 5 to about 3 during cultivation. At 24 h intervals 48 mg pepstatin A and 28 mg soy bean trypsin inhibitors (both from Sigma-Aldrich) were added to the cultures to minimize protein degradation. The culture supernatants were separated from the biomass by filtration through GF/A filters (Whatman). Protein expression levels were analyzed by RP-UPLC and were 0.2 g/L, 0.4 g/L, and 3.0 g/L for HFBI-DCBM-12, −24, and −48, respectively. The proteins were purified using aqueous two phase extraction and reverse-phase high-performance liquid chromatography (RP-HPLC) as described earlier35 (link) followed by lyophilization.

We collected water samples from multiple stations in each of the five Laurentian Great Lakes (Fig. 1A, Table S1) aboard the R/V Lake Guardian during the U.S. Environmental Protection Agency's Spring and Summer Survey cruises in 2012 and 2013. At each station, the CTD/rosette sampler generated temperature, fluorescence, dissolved oxygen and turbidity depth profiles as it descended through the water column and collected water samples from specific depths as it ascended to the surface. For every sampling event, we collected water from the surface (~2 m) and 10 m from the bottom, with the exception of comparatively shallow stations in Lake Erie (11–60 m) where bottom samples were collected 1 m from the bottom. During Summer Surveys, the water column of each lake was stratified and we collected samples from two additional depths: the DCL (if present) and the middle of the hypolimnion, an intermediate depth between the upper hypolimnion and bottom of the lake. Water samples (4.5–8 L) were pre‐filtered through GF/A filters (Whatman) and microorganisms were concentrated onto 0.22 μm Millipore Sterivex filters (SVGP01050) in 2012 and 0.22 μm Millipore Express Plus filters (GPWP04700) in 2013 using a peristaltic pump. Filters were stored at −80°C prior to DNA extraction.

Glass jars containing the sediment samples were covered with tin foil and freeze dried for 5 days. The sediment was thoroughly mixed and three subsamples of three replicates of 30 (± 2) g each were taken and transferred to individual Falcon tubes (n = 9, three per replicate). The remaining sediment was weighed.
Density separation was carried out by adding a saturated Sodium Iodide (NaI) solution (density = 1.79 g/cm³) to the Falcon tubes. The material was thoroughly mixed into suspension in the density solution and allowed to settle out completely. The supernatant was passed through a 75 µm sieve and the retained material (>75 µm) was transferred to an Erlenmeyer flask. This process was repeated a second time to enhance the recovery of microplastic from the sample. The >75 µm material from the first extraction was then subject to peroxidation using H₂O₂ to reduce the organic content. Briefly, 30 ml of 30% H₂O₂ was added to the sediment samples. An ice bath was prepared, and the Erlenmeyer flasks were placed in the ice bath if the reaction became too violent or if the temperature exceeded 40 °C. Temperatures higher than this can damage the microplastic. The samples were left in a laminar flow cabinet for 24 h, and the remaining material was vacuum filtered onto Whatman GF/A-filters. The >75 µm material from the second extraction was filtered directly onto GF/A-filters.