Nuclepore track etch

Conjugation was carried out using E. coli HB101 having pRL443 plasmid (Addgene) as described previously [65 ]. Twenty-five ml of SA7 or 6 ml of SA3 cultures of OD₇₃₀ of ~ 0.6 were centrifuged and washed twice with 1 ml of BG-11 medium. Likewise, 2 ml overnight grown cultures each of E. coli HB101 pRL443 and E. coli TOP10 containing the plasmid of interest (i.e., pAM3558_G and pAM3558_H) were centrifuged and washed twice with 100 µl milliQ water. The three pellets were then resuspended and mixed gently in 200 µl of BG-11 medium and the mixture was spread on a membrane (47 mm, 0.4 µm Nuclepore Track-Etch, Whatman, Maidstone, UK) placed on a BG-11 agar plate supplemented with 5% Luria Broth and incubated for 24 h at 38 °C and 100 µE light until mat cyanobacterial growth appears on the filter paper. Filter papers were then transferred to BG-11 plates having 10 µg ml⁻¹ of gentamicin and grown for 3 days at 38 °C and 100 µE light. Colony PCR was performed to select positive colonies, which were patched on higher antibiotic BG-11 plates to achieve complete chromosomal segregation.

Ten mL of the water sample was fixed immediately with 0.57 mL 35% formaldehyde solution (final concentration ∼2%) for 1 h at 4 °C in the dark. Fixed samples were then filtered on 0.2 µm polycarbonate filters (25 mm, Nuclepore Track-Etch; Whatman, Kent, UK) with 0.45 µm cellulose nitrate support filters (25 mm; Sartorius Stedim Biotech GmbH, Goettingen, Germany). The polycarbonate filters were air-dried and stored at −20 °C until further processing. Filters were mounted on microscope slides in DAPI-mix (final concentration 1 µg L⁻¹) and bacteria were counted using an epifluorescence microscope (×1,250). At least ten fields (each 0.0025 mm²) of a counting grid were counted per slide, or up to a minimum of 200 bacteria, when ten fields were not sufficient.

Phospholipid vesicles comprised of a molar fraction of 50% synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 30% chicken egg L-α-phosphatidylcholine (PC), and 20% porcine brain L-α- phosphotidylserine (PS) (Avanti Polar Lipids; Alabaster, AL) were prepared according to the methods of Shi et al., 2004 [12 (link)]. Phospholipids dissolved in chloroform were obtained from Avanti Polar Lipids (Alabaster, AL). 1.3 μmol DOPC, 0.78 μmol PC, and 0.52 μmol PS were added to dichloromethane (99.8%, Acros Organics, Morris Plains, NJ) in an acid-cleaned glass vial, and the solvent was evaporated under a gentle nitrogen stream while immersed in a 60°C water bath. Additional dichloromethane was added and evaporated from the vial three times to eliminate traces of chloroform. 1 mL of HBS (100 mMNaCl, 20mM HEPES, 0.02% w/v sodium azide, pH 7.5) was added to the dried phospholipids and vortexed until all phospholipids were dissolved, resulting in a uniform, cloudy solution. Liposomes were formed by extruding this solution through a double layer of polycarbonate membranes with 100 nm pore size (Nuclepore Track-Etch, Whatman, Maidstone, UK) 21 times using the Mini-Extruder extrusion device (Avanti Polar Lipids).