transfer in Fremellia diplosiphon, a blue-green alga with complementary adaptation
capabilities. Factors have been cloned to control F. diplosiphon’s complementary
adaptation capabilities at the transcription level.
To date, most conjugative transfer lines have used shuttle vectors with filamentous
blue-green algae and plasmids derived from those algae. Recently, conjugative
transfer with a wide range of host vector plasmids has been reported for the
single-celled blue-green alga Synechocystis PCC6803. In this line, the plasmid used
for conjugative transfer was pKT210, a wide-ranging vector plasmid from an Inc Q
population with replication units from RSF1010. This use of various vectors to
introduce genes through conjugation is expected to be a fruitful area in the future
(Dunahay et al. 1996; Radakovits et al. 2010; Larkum et al. 2012; Takeyama and
Matsunaga 1989).
7.4.3 Genetic Manipulation in Marine Blue-Green Algae
Genetically manipulation of marine blue-green algae is still in its early stages,
including the conjugative transfer method described below. The only examples
reported to date involve two species with natural transformation capabilities:
Agmenellum quadruplicatum PR6 and Synechococcus sp. PCC 7002. As with
freshwater blue-green algae, the gene to be introduced needs only to be mixed with
the cell to allow the cell’s absorption of the DNA and its combination with the
genome or replication of plasmids. Intrinsic plasmids have been discovered for A.
quadruplicatum PR6 and used with a shuttle vector line with an E. coli in an
attempt to express foreign proteins.
One example is an attempt to express pesticidal proteins produced by Bacillus
thuringiensis in A. quadruplicatum. To date, genetic manipulation in marine
blue-green algae has been restricted to the main strain. As marine biotechnology
develops in the future, replacement genetic manipulation techniques will have to be
developed quickly for screened marine blue-green algae with the ability to produce
useful materials or other special capabilities.
In Japan, Matsunaga et al. attempted to develop a genetically manipulated
variety of the marine blue-green alga Synechococcus sp. NKBG042902. A preliminary examination of plasmids that could be used for transformation line shuttle
vectors for this strain showed four types: pSY08 (>10 kb), pSY09 (around 10 kb),
pSY10 (2.7 kb), and pSY11 (2.3 kb). Among these, pSY11 was used to create a
shuttle vector with E. coli, as it has a relatively high copy number and low
molecular weight. pSY’s restriction enzyme sites are shown in Fig. 7.5. As a
plasmid, its copy number is high at 30–50 per genome. A 1.4 kb Hind III fragment
from pSY18 has been introduced at a pUC18 multicloning site to form the hybrid
plasmid pUSY02 (Moore et al. 1988; Takeyama and Matsunaga 1989).
As a host, Synechococcus sp. NKBG042902 possesses pSY 11 , raising concerns
that the shuttle vector may be excluded due to incompatibility. To address this, the
curing strain 042902-YG1116 was created for the same strain. This curing strain is
obtained by treating the 042902 strain with acridine orange for random selection
and cloning, and does not include pSY 10 or pSY 11 (Matsunaga 1992).
7.4 Biotechnology of Microalgae
213
capabilities. Factors have been cloned to control F. diplosiphon’s complementary
adaptation capabilities at the transcription level.
To date, most conjugative transfer lines have used shuttle vectors with filamentous
blue-green algae and plasmids derived from those algae. Recently, conjugative
transfer with a wide range of host vector plasmids has been reported for the
single-celled blue-green alga Synechocystis PCC6803. In this line, the plasmid used
for conjugative transfer was pKT210, a wide-ranging vector plasmid from an Inc Q
population with replication units from RSF1010. This use of various vectors to
introduce genes through conjugation is expected to be a fruitful area in the future
(Dunahay et al. 1996; Radakovits et al. 2010; Larkum et al. 2012; Takeyama and
Matsunaga 1989).
7.4.3 Genetic Manipulation in Marine Blue-Green Algae
Genetically manipulation of marine blue-green algae is still in its early stages,
including the conjugative transfer method described below. The only examples
reported to date involve two species with natural transformation capabilities:
Agmenellum quadruplicatum PR6 and Synechococcus sp. PCC 7002. As with
freshwater blue-green algae, the gene to be introduced needs only to be mixed with
the cell to allow the cell’s absorption of the DNA and its combination with the
genome or replication of plasmids. Intrinsic plasmids have been discovered for A.
quadruplicatum PR6 and used with a shuttle vector line with an E. coli in an
attempt to express foreign proteins.
One example is an attempt to express pesticidal proteins produced by Bacillus
thuringiensis in A. quadruplicatum. To date, genetic manipulation in marine
blue-green algae has been restricted to the main strain. As marine biotechnology
develops in the future, replacement genetic manipulation techniques will have to be
developed quickly for screened marine blue-green algae with the ability to produce
useful materials or other special capabilities.
In Japan, Matsunaga et al. attempted to develop a genetically manipulated
variety of the marine blue-green alga Synechococcus sp. NKBG042902. A preliminary examination of plasmids that could be used for transformation line shuttle
vectors for this strain showed four types: pSY08 (>10 kb), pSY09 (around 10 kb),
pSY10 (2.7 kb), and pSY11 (2.3 kb). Among these, pSY11 was used to create a
shuttle vector with E. coli, as it has a relatively high copy number and low
molecular weight. pSY’s restriction enzyme sites are shown in Fig. 7.5. As a
plasmid, its copy number is high at 30–50 per genome. A 1.4 kb Hind III fragment
from pSY18 has been introduced at a pUC18 multicloning site to form the hybrid
plasmid pUSY02 (Moore et al. 1988; Takeyama and Matsunaga 1989).
As a host, Synechococcus sp. NKBG042902 possesses pSY 11 , raising concerns
that the shuttle vector may be excluded due to incompatibility. To address this, the
curing strain 042902-YG1116 was created for the same strain. This curing strain is
obtained by treating the 042902 strain with acridine orange for random selection
and cloning, and does not include pSY 10 or pSY 11 (Matsunaga 1992).
7.4 Biotechnology of Microalgae
213