How are K. lactis strains stored for the long term?
K. lactis cells can be stored at –70°C suspended in a final concentration of 20% (v/v) sterile glycerol. For example, 500 µl of a culture of freshly grown cells can be diluted with 500 µl of a sterile 40% glycerol solution (to give a 20% final glycerol concentration). Alternatively, a scoop of cells from a freshly grown streak on agar medium can be scraped from the plate using a sterile loop or pipet tip and resuspended directly in 20% sterile glycerol. It is important to make sure that all solutions and tubes that contact the cells are sterilized prior to their use. To revive frozen K. lactis cells containing an integrated pKLAC1 construct, streak a small aliquot of frozen cells on YCB agar medium supplemented with 5 mM acetamide. After this initial growth on YCB agar medium, the cells can be grown without selection in rich medium for protein expression.
