Method for non-vector modification of eukaryotic unicellular algae using a mixture of colchicine and cytochalasin B to increase the cell size

摘要

The notified method is the vector-free modification method of eukaryotic monocellular algae, which is characterised by: is subjected to 24-hour induction of changes in the nuclear genome and cytoplasmic microalgae cells using two simultaneous compounds: colchicine (microtubular polymerisation inhibitor) at the concentration of 0.00125% to 0.01% and cytochalazine B (active polymerisation inhibitor) at the concentration of 0.002 mg/mL to 0.004 mg/mL and then remove the inhibitors from the substrate and select the cells according to the following sequential cycles,where the first cycle is cultivated for a period of three to seven days in a 5 mL medium without colchicine and cytochalazine B at a temperature of 25 to 30,C with continuous blue light in the range of 460 nm-465 nm and red in the range of 625 nm-640 nm on a shaken 6-hole plate, the following cycle is cultivated for a period of between seven and fourteen days in the volume of 250 mL in a growing medium exchanged at least once every three or four days at the temperature 25-30oC with continuous blue light in the range 460 nm-465 nm and red light in the range of625 nm -640 nm. The next cycle is to be grown for a period of five to seven days in a photobioreactor of the volume of min. 2.5L at temperature from 28 to 35oC with continuous blue light within 460 nm-465 nm and red within 625 nm-640 nmIt is advantageous when, during the last stage of the selection cycle, the medium is transferred with the cells to the container at temperature 4,C for a 24-hour period, then decontamination and transfer the cells to a fresh medium.