phase of DNA, which can easily integrate foreign genes, and
the cell activity is strong. The cells recover quickly after electric
shock.
5. Osmotic treatment is helpful to improve the electroporation
efficiency of Chlorella spp. The reason may be that the cell
membrane is perforated and the cytoplasm can easily flow out
during electroporation, which leads to cell death. After hyperosmotic treatment, cells are in a dehydrated state, which can
reduce the loss of cytoplasm caused by cell membrane
perforation.
6. Ice bath after electric shock prolongs the refolding time of
perforated cell membranes, helps to increase gene intake, but
also increases cell mortality.
7. A certain number of algal cells have a limited ability to absorb
plasmids during electric shock. A low dose of plasmids lead to a
lower transformation rate. An excessive dose of plasmids may
inhibit each other to reduce the transformation rate, so there is
a suitable range of plasmid concentration.
8. Chlorella cells have strong nuclease activity, and exogenous
genes can easily be degraded after transfection. Therefore,
adding a certain concentration of salmon sperm DNA during
electroporation can competitively bind to the nuclease in algal
cells, thereby protecting the plasmid and improving the transformation efficiency.
9. The efficiency of electroporation is directly proportional to the
product of pulse field strength and width, and there is a complementary relationship between them.
10. Pulse field intensity is the most important parameter in the
process of electrotransformation. If the intensity is not enough,
the cell will not reach the critical voltage of perforation. If the
intensity is too high, the cell will crack and die because of a
large number of perforations. In order to achieve better transformation efficiency, it is usually necessary to use a higher pulse
intensity than the critical permeabilizing field strength, which
will cause some algal cells to lyse and die. Previous studies
found that the transformation efficiency of Chlamydomonas
reinhardtii was optimal when the cell death rate was 50%
[9, 10].
Due to the nonuniform size of algal cells, the critical field
intensity will be different. Under the optimum pulse field
intensity, some algal cell membranes will crack and die because
of irreversible breakdown. Some algal cells cannot form perforation. We need to control the optimal ratio of algal cells with
reversible perforation.
184
Liang Ji and Jianhua Fan
the cell activity is strong. The cells recover quickly after electric
shock.
5. Osmotic treatment is helpful to improve the electroporation
efficiency of Chlorella spp. The reason may be that the cell
membrane is perforated and the cytoplasm can easily flow out
during electroporation, which leads to cell death. After hyperosmotic treatment, cells are in a dehydrated state, which can
reduce the loss of cytoplasm caused by cell membrane
perforation.
6. Ice bath after electric shock prolongs the refolding time of
perforated cell membranes, helps to increase gene intake, but
also increases cell mortality.
7. A certain number of algal cells have a limited ability to absorb
plasmids during electric shock. A low dose of plasmids lead to a
lower transformation rate. An excessive dose of plasmids may
inhibit each other to reduce the transformation rate, so there is
a suitable range of plasmid concentration.
8. Chlorella cells have strong nuclease activity, and exogenous
genes can easily be degraded after transfection. Therefore,
adding a certain concentration of salmon sperm DNA during
electroporation can competitively bind to the nuclease in algal
cells, thereby protecting the plasmid and improving the transformation efficiency.
9. The efficiency of electroporation is directly proportional to the
product of pulse field strength and width, and there is a complementary relationship between them.
10. Pulse field intensity is the most important parameter in the
process of electrotransformation. If the intensity is not enough,
the cell will not reach the critical voltage of perforation. If the
intensity is too high, the cell will crack and die because of a
large number of perforations. In order to achieve better transformation efficiency, it is usually necessary to use a higher pulse
intensity than the critical permeabilizing field strength, which
will cause some algal cells to lyse and die. Previous studies
found that the transformation efficiency of Chlamydomonas
reinhardtii was optimal when the cell death rate was 50%
[9, 10].
Due to the nonuniform size of algal cells, the critical field
intensity will be different. Under the optimum pulse field
intensity, some algal cell membranes will crack and die because
of irreversible breakdown. Some algal cells cannot form perforation. We need to control the optimal ratio of algal cells with
reversible perforation.
184
Liang Ji and Jianhua Fan