TITLE: Transformation of Synechocystis sp. PCC 6803
AUTHORS: Anika Wiegard, Ilka Maria Axmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to transform naturally competent </span><span style = "font-style:italic;">Synechocystis</span><span> sp. PCC 6803.</span></div><div class = "text-block"><span>Use this protocol, if you aim at integrating a nucleotide sequence in the </span><span style = "font-style:italic;">Synechocystis</span><span> chromosome via homologous recombination. Be aware that </span><span style = "font-style:italic;">Synechocystis </span><span>PCC 6803 strains from different labs vary in their transformation efficiency.</span></div><div class = "text-block">The protocol was modified from</div><div class = "text-block"><span>Grigorieva, G. & Shestakov, S. (1982). Transformation in the cyanobacterium </span><span style = "font-style:italic;">Synechocystis </span><span>sp. 6803. FEMS Microbiol Lett 13, 367-370.</span></div></div>

[STEPS]
?. Grow Synechocystis sp. PCC 6803 cells in BG-11 medium (see e.g. Köbler and Wilde https://dx.doi.org/10.17504/protocols.io.wj5fcq6 for recipe) until OD750 nm =~0.5 -1
should take about 4 days after inoculation
?. Transfer 10 ml of the cell suspension to a sterile 15 ml falcon tube and spin down at ~ 2 200 rcf at room temperature.
?. Discard supernatant, resuspend the pellet in a small volume of residual supernatant (~200-500 µl) and transfer the cells to a sterile 1.5 ml tube (handle cells carefully, don’t pipet up and down too often, use large pipet tips to minimize shear forces).
handle cells carefully, don’t pipet up and down too often, use large pipet tips to minimize shear forces
?. Add 5  µl plasmid DNA to the cells
for 'easy' transformations DNA amount seems not to be critical, but 2 µg should be fine
?. Incubate for 30 min at room temperature (in the tube) without shaking.
?. Plate on a very thick BG-11 plate (1 % Bacto agar, in 1x BG-11) without antibiotics (~ 50 ml BG-11 agar per plate).
?. Wait for a few hours until residual liquids are absorbed by the agar (in laminar flow hood).
?. Underlay with antibiotics to create a gradient in the plate:using a sterile spatula lift the agar at one end of the plateKan: add 300-400 µl of 1mg/ml kanamycin stock solution under the agar at the end of the plateCm: incubate the plate for 2-3 days without antibiotics, then add 400 µl of 1.4 mg/ml chloramphenicol stock solution under the agar at one end of the plateplace agar back in placeantibiotic will diffuse trough the plate forming a gradient
?. Incubate at low light intensities (~40 μmol Photonen m-2 s-1, optional: cover the plate with a thin sheet of white paper) and 30 °C until first (isolated) green colonies appear (lid facing upwards!!!)
?. When first colonies appear, pick them (only green ones) and streak on a plate with slightly higher antibiotic concentration (for km: e. g. 2 or 4 µg/ml, for Cm: e.g. 1.5 or 2.5 µg/ml). In the beginning, don’t streak them over the whole plate, but streak small spots. Incubate at ~ 60 µmol photons m-2 s-1, facing the lid down.
?. Increase concentration of antibiotic stepwise (e.g. Kan: 4–8-12-20-40 or more µg/ml, Cm: 2.5-5-7-14 µg/ml etc) until full segregation is achieved (check by colony PCR and/or southern blot).