Vswp01300

Both the insert and step 1 sgRNA vector were digested with BsmBI for 3 h at 55 °C and subsequently purified via a Qiaquick PCR Purification column. The ligation reactions were then set up using 100 ng of vector, 100 ng of insert, 2 μL of buffer, 1 μL of T4 ligase (NEB M0202T), and ultra pure H₂O up to 20 μL. Each reaction was allowed to proceed overnight at 16 °C. The following morning the ligase was heat inactivated at 65 °C for 20 min. Following this, the reaction was dialyzed into ultrapure water (Millipore VSWP01300) to remove any residual salts from the ligase buffer. Once the DNA was dialyzed, the ligation reaction was split evenly between 300 μL of Stbl4 electrocompetent cells, which were then transformed according to the manufacturer's protocol. The transformed cells were resuspended in 10 mL of SOC media (Invitrogen 15544034) and allowed to recover for 1 h shaking before being used to inoculate 150 mL of LB media supplemented with carbenicillin. After 16 h of further growth, plasmid DNA containing the sgRNA library was isolated via a Qiagen Plasmid Plus MaxiPrep kit (Qiagen 12963).

ADP1-ISx glycerol stocks were streaked out on LB agar and incubated overnight. For each transformation assay replicate, a different colony was picked and inoculated into LB and grown overnight to produce a culture ready for transformation. Transformations in liquid culture were initiated by mixing input DNA, 250 μl LB, and 17.5 μl of the overnight ADP1-ISx culture. Puddle transformations were initiated by mixing input DNA with 50 μl of the overnight ADP1-ISx culture and transferring the complete volume onto a 13 mm diameter, 0.025 μm pore-size mixed cellulose esters membrane (Millipore #VSWP01300) that was left resting on the surface of an LB agar plate. In both cases, transformations were incubated for 24 h under normal A. baylyi growth conditions. Then, dilutions in sterile saline were plated on LB and LB-Kan agar to estimate transformation frequencies or on LB-Kan or LB-AZT agar for genome modification steps.

Hfx. volcanii was grown in Erlenmeyer flasks in synthetic medium with casamino acids (0.25% (w/v)) as carbon and energy source in the presence of 100 µg/ml genomic DNA as phosphate source. The culture was started with an OD₆₀₀ of 0.1 and grown into stationary phase. At various time points 1 ml aliquots were removed and cells were removed by centrifugation (8000 g, 5 min, room temperature). 30 µl aliquots of the supernatants were dialyzed on membrane filters (Millipore, 13 mm diameter, VSWP01300) against distilled water and analyzed by analytical agarose gel electrophoresis.