"The transformation cassette was prepared by digestion of plasmid pMJ013b (GenBank accession number KJ572788) with MlyI (New England Biolabs) at 37°C for 1 h. Digestion products were separated on a 1% (w/v) agarose gel, and the 2660-bp digested DNA fragment was extracted using a QIAquick gel purification kit (Qiagen), according to the manufacturer’s instructions. The purified fragment was analyzed with an Agilent Bioanalyzer (Supplemental Figure 8) and used for transformation.
CMJ030 was grown in TAP medium in a 20-liter container under 100 µmol photons m^(–2) s^(–1) cool white fluorescent light, with continuous stirring and bubbled air, until it reached a cell density of 1.5 x 10^6 cells/mL. Cells were collected as follows. Bubbling was stopped and the 20-liter container was transferred into a 32-gallon garbage bin and illuminated from the top by four cool white fluorescent bulbs for 2 h. This caused the cells to settle to the bottom of the 20-liter container. The top 15 liters were removed by aspiration, and the lower 5 liters were centrifuged in RC5C centrifuges (Sorvall Instruments) with GS3 rotors for 4 min at 1000g. Pellets were resuspended in TAP supplemented with 40 mM sucrose at 2 x 10^8 cells/mL. Transformation was performed by electroporation according to Shimogawara et al. (1998) with some modifications. Transforming DNA (144 µL) at 52 ng/µL was added to a sterile 50-mL Falcon tube with 50 mL of concentrated cells (37.5 ng DNA per 250 µL concentrated cells) in 40 mM sucrose. The concentrated cells were incubated with transforming DNA at 16°C for at least 80 min before electroporation [slight modification of method from Zhang et al., 2014, where this step was 20 min]. The cell/DNA mix was then aliquoted into sterile electroporation cuvettes (4-mm gap, 1.5-mL Micro Cuvette, two Clear Sides, E&K Scientific) at 250 µL/cuvette. Cells were electroporated (Bio-Rad; Gene Pulser2 electroporation system) with pulse settings of 800 V and 25 µF, followed by immediate decanting into a 15-mL Falcon tube containing 13 mL of TAP supplemented with 40 mM sucrose. The 15-mL Falcon tubes were shaken gently under low light (5 µmol photons m^(–2) s^(–1)) for 6 h. Cells were then collected by centrifugation at 1000g for 4 min, most of the supernatant was decanted, and the cells were resuspended in the remaining 500 µL of supernatant. Resuspended cells were gently plated onto 2% (w/v) TAP agar plates containing 20 µg/mL paromomycin. These plates were stored at 5 µmol photons m^(–2) s^(–1) light for 2 weeks, until transformant colonies appeared."
Li X*, Zhang R*, Patena W*, Gang SS, Blum SR, Ivanova N, Yue R, Robertson JM, Lefebvre PA, Fitz-Gibbon ST, Grossman AR, Jonikas MC (2016)
The Plant Cell.