allowed transformation to take place without plant cells being subjected to protoplast formation, and DNA can also be introduced into organelles within the cell.
Matsunaga et al. have attempted to apply the microprojectile approach to
increase transformation efficiency and manipulability in blue-green algae. While
particles of tungsten and gold have been used thus far as DNA carriers in the
microprojectile method, they are relatively large, with a diameter of around 0.1–
0.5 lm that has rendered them unsuitable for transformation in prokaryotes.
Application research has been carried out on magnetic particles synthesized with
the cells of magnetotactic bacteria. These magnetic bacterial particles are very small
(around 50 nm) and even and are covered with an evenly surfaced organic membrane that allows for easy chemical modification. This has resulted in explorations
of the applicability of magnetic bacterial particles as DNA carriers.
Modification of magnetic bacterial particles with c-APTES, glutaraldehyde, and
triamine allows for adsorption and fixing of over twice as much DNA as the carriers
previously used with particles with a DNA fixation weight of 2 lg/mg. Transformation with magnetic bacterial particle DNA carriers through the microprojectile
approach using highly pressurized air in the marine blue-green alga Synechococcus
sp. and the freshwater blue-green alga Anabaena sp. PCC 7120 showed a cellular
magnetic response of up to 1.5% in marine blue-green algae and 7.5% in freshwater
blue-green algae when sprayed at a pressure of 100 kgf/cm
2 , indicate a very high
efficiency of DNA carrier introduction within the cell. Although the respective
shuttle vectors pUSY02 and pRL5 were mixed to the DNA carrier in the marine and
freshwater blue-green algae in this experiment, transformation could be achieved
under these conditions. Observation of these magnetic responsive cells with a
transmission electron microscope showed DNA carriers to be present near the
membrane in both.
In the future, transformation is expected to be made possible through increases in
the DNA carrier spraying speed. Many marine blue-green algae have surfaces
sheathed in polysaccharides or cells that are rendered incapable of agglutination or
growth when washed. For this reason, the microprojectile approach appears to be a
promising method for application with marine blue-green algae that cannot be
simply transformed.
An interesting property was recently reported in pSY10, one of the plasmids for
the marine blue-green alga Synechococcus sp. As mentioned, the shuttle vector
PUSY02 was developed from the same strain’s high-replication plasmid pSY11.
While a defined copy number has been identified for pSY11 regardless of culturing
conditions, pSY10 shows the same copy number as pSY11 under normal culturing
conditions, with a copy number that increased sharply according to those conditions. While pSY10’s copy number is 30–50 per genome (similar to pSY11) when
the strain is cultured beforehand in a medium with low saline concentration, it rises
sharply when transferred to a medium with a high saline concentration, reaching
150 under a concentration of 0.5–2% and around 250 under a concentration of 3%
within 20 h (Fig. 7.6).
This phenomenon has not been observed at all in three other plasmids from the
same strain, including pSY11. The rise in pSY10’s copy number was also not
7.4 Biotechnology of Microalgae
215
Matsunaga et al. have attempted to apply the microprojectile approach to
increase transformation efficiency and manipulability in blue-green algae. While
particles of tungsten and gold have been used thus far as DNA carriers in the
microprojectile method, they are relatively large, with a diameter of around 0.1–
0.5 lm that has rendered them unsuitable for transformation in prokaryotes.
Application research has been carried out on magnetic particles synthesized with
the cells of magnetotactic bacteria. These magnetic bacterial particles are very small
(around 50 nm) and even and are covered with an evenly surfaced organic membrane that allows for easy chemical modification. This has resulted in explorations
of the applicability of magnetic bacterial particles as DNA carriers.
Modification of magnetic bacterial particles with c-APTES, glutaraldehyde, and
triamine allows for adsorption and fixing of over twice as much DNA as the carriers
previously used with particles with a DNA fixation weight of 2 lg/mg. Transformation with magnetic bacterial particle DNA carriers through the microprojectile
approach using highly pressurized air in the marine blue-green alga Synechococcus
sp. and the freshwater blue-green alga Anabaena sp. PCC 7120 showed a cellular
magnetic response of up to 1.5% in marine blue-green algae and 7.5% in freshwater
blue-green algae when sprayed at a pressure of 100 kgf/cm
2 , indicate a very high
efficiency of DNA carrier introduction within the cell. Although the respective
shuttle vectors pUSY02 and pRL5 were mixed to the DNA carrier in the marine and
freshwater blue-green algae in this experiment, transformation could be achieved
under these conditions. Observation of these magnetic responsive cells with a
transmission electron microscope showed DNA carriers to be present near the
membrane in both.
In the future, transformation is expected to be made possible through increases in
the DNA carrier spraying speed. Many marine blue-green algae have surfaces
sheathed in polysaccharides or cells that are rendered incapable of agglutination or
growth when washed. For this reason, the microprojectile approach appears to be a
promising method for application with marine blue-green algae that cannot be
simply transformed.
An interesting property was recently reported in pSY10, one of the plasmids for
the marine blue-green alga Synechococcus sp. As mentioned, the shuttle vector
PUSY02 was developed from the same strain’s high-replication plasmid pSY11.
While a defined copy number has been identified for pSY11 regardless of culturing
conditions, pSY10 shows the same copy number as pSY11 under normal culturing
conditions, with a copy number that increased sharply according to those conditions. While pSY10’s copy number is 30–50 per genome (similar to pSY11) when
the strain is cultured beforehand in a medium with low saline concentration, it rises
sharply when transferred to a medium with a high saline concentration, reaching
150 under a concentration of 0.5–2% and around 250 under a concentration of 3%
within 20 h (Fig. 7.6).
This phenomenon has not been observed at all in three other plasmids from the
same strain, including pSY11. The rise in pSY10’s copy number was also not
7.4 Biotechnology of Microalgae
215