Potassium Citrate

MosSCI transformation was performed based on the protocol described in [3] (link)
(http://sites.google.com/site/jorgensenmossci/). The Mos1
insertion strains EG4322 or EG5003 were used for injection. Injection mixes
contained pJL43.1 (50 ng/µl), pCJF90 (2.5 ng/µl), pCFJ104 (5
ng/µl), and the respective expression clone (50 ng/µl) in 20 mM
potassium phosphate and 3 mM potassium citrate (pH 7.5). We note that although
we were able to obtain transgenic strains expressing each of the constructs
described, some apparent integration events did not result in detectable
expression; we do not know the reason for this variability.

Viruses were resuspended using a previously optimized method for these soils (Trubl et al., 2016 (link)) with minor adjustments. Briefly, 10 ml of a 1% potassium citrate resuspension buffer amended with 10% phosphate buffered-saline and 150 mM magnesium sulfate was added to 10 ± 0.5 g peat (AKC’ buffer). Viruses were physically dispersed via 1 min of vortexing, 30 s of manual shaking, and then 15 min of shaking at 400 rpm at 4 ° C. The samples were then centrifuged for 20 min at 1, 500 ×g at 4 °C to pellet debris, and the supernatant was transferred to new tubes. The resuspension steps above were repeated two more times and the supernatants were combined, and then filtered through a 0.2 µm polyethersulfone membrane filter to remove particles and cells and transferred into a new 50 ml tube. The filtrate was then purified via overnight treatment with DNase I (Kunitz units; ThermoFisher, Waltham, Massachusetts) at a 1:10 dilution at 4 °C, inactivated by adding a final concentration of 10 mM EDTA and EGTA and mixing for 1 h. All viral particles were further purified by CsCl density gradients, established with five CsCl density layers of ρ 1.2, 1.3, 1.4, 1.5, and 1.65 g/cm³; we included a 1.3 g/cm³ CsCl layer to collect ssDNA viruses (Thurber et al., 2009 (link)). After density gradient centrifugation of the viral particles, we collected and pooled the 1.3–1.52 g/cm³ range from the gradient for viral DNA extraction. The viral DNA was extracted (same elution volume) using one of three methods: Wizard mini columns (Wizard; Promega, Madison, WI, products A7181 and A7211), cetyl trimethylammonium bromide (CTAB; Porebski, Bailey & Baum, 1997 (link)), or modified DNeasy PowerSoil DNA extraction kit (C3 reagent was 1/3 of working volume and C4 reagent was 1.5 × working volume) with heat lysis (10 min incubation at 70 °C, vortexing for 5 s, and 5 min more of incubation at 70 °C) (PowerSoil; Qiagen, Hilden, Germany, product 12888). The extracted DNA was further cleaned up with AMPure beads (Beckman Coulter, Brea, CA, product A63881). DNA purity was assessed with a Nanodrop 8000 spectrophotometer (Implen GmbH, Germany) by the reading of A260/A280 and A260/A230, and quantified using a Qubit 3.0 fluorometer (Invitrogen, Waltham, Massachusetts). DNA sequencing libraries were prepared using Swift Accel-NGS 1S Plus DNA Library Kit (Swift BioSciences, Washtenaw County, Michigan), and libraries were determined to be ‘successful’ if there was a smooth peak on the Bioanalyzer with average fragment size of <1kb (200–800 bp ideal) and minimal-to-no secondary peak at ∼200 bp (representing concatenated adapters) (Fig. S1), and <20 PCR cycles were required for sequencing. Six libraries were successful (two from bog and four from fen) and required 15 PCR cycles. The successful libraries were sequenced using Illumina HiSeq (300 million reads, 2 × 100 bp paired-end) at JP Sulzberger Columbia Genome Center.

Several beverage formulations were initially developed in order to reach the effective concentration of the compounds, while sustaining the solubility and the desired organoleptic characteristics of the beverage, using laboratory scale trials involving staff and students in the food lab as panelists to evaluate the formulations. The final beverage formulation contains citrulline malate (2.4% w/v), magnesium citrate (0.4% w/v), tripotassium citrate (0.4% w/v), trisodium citrate (0.4% w/v), mixed berries powder (0.3% w/v), rosehip powder (0.2% w/v), cocoa extract (0.2% w/v), caffeine anhydrous (0.05% w/v), steviol glycosides (3% w/v), and maltodextrin (0.2% w/v). This formulation was in the form of an instant beverage; viz., in the form of powder that is dissolved in water. Specifically, one serving of the final beverage is equal to 23.35 g of the powder dissolved in 250 mL of water.

Food-grade citrulline malate 2:1 (THG plc., Manchester, UK), magnesium citrate (THG plc., Manchester, UK) tripotassium citrate (Manis Chemicals, Athens, Greece), trisodium citrate (Manis Chemicals, Athens, Greece), cocoa extract (Foodspring GmbH, Berlin, Germany), rosehip powder (Health Trade O.E., Achaia, Greece), mixed frozen berries (blueberries 20%, strawberries 20%, raspberries 20%, black gooseberries 20%, red gooseberries 20%; Lidl Hellas & SIA OE., Athens, Greece), maltodextrin (Manis Chemicals, Athens, Greece), caffeine anhydrous (Manis Chemicals, Athens, Greece), and steviol glycosides (Only Bio, Athens, Greece) were bought and used to prepare beverage samples.