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Spheroplast generation of E. coli

FW Felix Weihs
KW Katarzyna Wacnik
RT Robert D. Turner
SC Siân Culley
RH Ricardo Henriques
SF Simon J. Foster
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Overnight-cultures of E. coli C43(DE3) mreD-eyfp (E. coli C43(DE3) containing the plasmid pWALDO-mreD-eyfp) were diluted in BHI to an OD600 = 0.05 and grown at 37 °C for 1 h in the presence of kanamycin (50 µg/ml), 250 rpm. Subsequently, 0.6 mM IPTG was added and cultures were grown for another 2 h at 37 °C. Cells were harvested and washed with ice-cold Tris-HCl (10 mM, pH 7.5) and the pellet was resuspended to an OD600 = 0.6 in sucrose buffer (33 mM Tris-HCl (pH 8.0), 20% sucrose (w/v)). 80 µl of EDTA (0.1 M) and 400 µl lysozyme (1 mg/ml) were added to 1 ml of resuspended cells and the tube was covered in foil and incubated at 4 °C on a rotary wheel for 30 min. Cells were then washed with and resuspended in ice-cold sucrose buffer. Prepared spheroplasts were placed on non-coated glass slides and analysed by fluorescence microscopy.

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