The cells were then concentrated by centrifugation and diluted to a concentration of 50–100 μg Chl a/ml. 10 μg plasmid DNA
dissolved in sterile distilled water were added to ice-cooled microcentrifuge tubes followed EPZ-6438 in vivo by 40 μl of concentrated cell culture. The cooled cell suspensions were transferred to an ice-cooled electroporation cuvette (2-mm electrode gap, Eppendorf) and exposed to a single electrical pulse. The pulse was delivered by a Gene-Pulser Xcell Microbial System (Bio-Rad Laboratories) set at 25 μF, 300 Ω and 1.6 kV. Immediately following the discharge, the suspensions were cooled on ice for about 5 min and thereafter transferred to culture flasks, containing ammonium supplemented growth medium, and left over night to recover. The cells were harvested and plated on ammonium supplemented, ampicillin containing plates. The plates were kept at low illumination (2–3 μmol of photons m-2 s-1) and after 2 to 3 weeks of selection, positive colonies were picked
and transferred to liquid medium supplemented with ammonium and ampicillin as detailed above. When the colonies had adjusted to the transition from growing on plates to liquid medium they were kept at standard illumination and transferred to plain growth medium to develop heterocysts. The constructs in the transformed GDC-0973 research buy cultures were confirmed by colony PCR. The primers used for the colony PCR (pSUN202 seq primer forward and reverse) anneal to the vector sequences flanking the inserted promoter region and hence the product spans the full length of the insert (Table 1). Fluorescence and luminescence measurements Fluorescence emission of GFP was measured from whole cells (100 μl N. punctiforme culture at a concentration of 30 μg Chl a/ml) with an excitation wavelength of 488 nm and an emission wavelength of 520 nm using a Molecular Imager PharosFX Plus (Bio-Rad Laboratories). Luminescence from luciferase activity was induced by the addition
of the substrate Decanal Phospholipase D1 (n-Decyl Aldehyde, Sigma) to the cyanobacterial suspension. To 100 μl N. punctiforme culture (at a concentration of 30 μg Chl a/ml) 5 μl of a Decanal mixture was added. The mixture consisted of 7.8 μl Decanal, 500 μl Methanol (Fluka) and 500 μl distilled water. Light emission was monitored with a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories). Fluorescence and luminescence measurements were performed at room temperature. Measurement data was corrected to the background (cells containing empty vector) and normalized to the chlorophyll a concentration of the samples. All measurements within one experiment were made in triplicate and performed at least three times using two independent clones. The clones containing the constructs pPprbcL-gfp and pPprbcL-lux were used as positive controls. Localization of GFP fluorescence was viewed in a fluorescence microscope (Leica DMRXE, Leica microsystems) with an excitation wavelength of 460–500 nm and an emission wavelength of 512–542 nm.