Cryopreservation of Synechococcus elongatus UTEX 2973
The rapidly growing cyanobacterium Synechococcus elongatus UTEX 2973 is an ideal candidate for genetic modification. A cryopreservation protocol suitable for this strain was developed by optimizing three main parameters: cryoprotectant type and concentration, cell growth phase, and freezing procedure. Cells harvested in stationary phase were more viable after cryopreservation compared to log phase and > 80% viability could be obtained by any one of the three freezing procedures tested by using 1% (v/v) dimethyl sulfoxide (DMSO) as a cryoprotectant. Application of the optimized protocol with a freezing procedure which controlled the rate of cooling to − 1 °C min−1 demonstrated that the presence of genetic modifications resulted in reduced cell viability after cryopreservation. Cryopreservation of knockout mutant strains indicated that the deletion of either the glucose-1-phosphate adenylyltransferase (glgC) gene or the acyl-ACP synthetase (aas) gene reduced viability by 30 or 50%, respectively. Synechococcus elongatus UTEX 2973 could be cryopreserved up to 6 months and recovered either in liquid or solid media with no significant reduction in viability.
KeywordsCyanobacteria Cryopreservation Cryoprotectant Synechococcus elongatus 2973 Glycogen CRISPR-Cas9
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