Note: Descriptions are shown in the official language in which they were submitted.
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BIOSYNTHESIS OF FOREIGN PROTEINS USING
TRANSFORMED MICROALGAE
FIELD OF THE INVENTION
1. FILED OF THE INVENTION
The present invention relates to a method for biosynthesis of foreign protein
using transformed microalgae. More particularly, it relates to a method for
biosynthesis of foreign proteins by transforming microalgae protoplasts with
DNA
vector containing intended foreign protein gene, and then culturing it in a
large scale.
2. DESCRIPTION OF THE PRIOR ARTS
Escherichia coli is the most widely used heterologous expression system, but
the bacterium has some limitations including; i) poor or no expression of
certain
proteins, ii) some recombinant proteins lack biological activity, iii) some
recombinant proteins are toxic to Escherichia coli, and iv) some recombinant
proteins form insoluble inclusion bodies. Similar problems can occur with
yeast
expression systems. Cultured mammalian and insect cells have been used to
solve
these problems, but these systems can be expensive because of the cost of
media,
2o equipment and requirement for extensive purification procedures.
Therefore, the present inventors made an investigation on chlorella
transformation as a new heterologous overexpression system, which could
substitute
for Escherichia coli, to solve the above-described problems.
As a result thereof, we have found that microalgae expression system was
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more economical than cell culture or animal or plant expression system because
the
microalgae had simpler metabolic pathway than those of animals or plants had
and
could be cultured in a large scale using an aquarium with light and carbon
dioxide.
Moreover, the fact that microalgae has post-translational modification process
unlike
Escherichia coli indicates that the biological activity of foreign protein
expressed in
microalgae should be more similar to that of naturally occurnng protein. Under
this circumstance, we intended to develop microalgae overexpression system for
producing foreign proteins.
There had been attempts to transform the Chlorella species, one of
1o microalgae. Jarvis and Brown described the transient expression of
luciferase in
protoplasts of Chlorella ellipsoidea (Jarvis, E. E., and Brown, L. M. 1991.
Transient
expression of firefly luciferase in protoplasts of the green alga Chlorella
ellipsoidea,
Current Genetics 19, 317-321) and Dawson et al. found that nitrate reductase-
deficient mutants of Chlorella sorokiniana could be rescued by transforming
them
with nitrate reductase gene isolated from Chlorella vulgaris(Dawson, H. N.,
Burlingame, R., and Cannons, A. C. 1997. Stable transformation of Chlorella:
Rescue of nitrate reductase-deficient mutants with the nitrate reductase gene.
Current Microbiology 35, 356-362). However, these experiments described only
transient expression or expression of protein genes originated from chlorella
species.
Therefore, the present inventors made investigations on a method for
biosynthesizing foreign protein by transforming protoplasts of microalgae with
a
vector DNA containing the genes originated from organisms other than
microalgae
and then culturing them in a large scale. As a result thereof, we found out
that
objects described above could be reached with this method.
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SUMMARY OF THE INVENTION
The object of present invention is to provide a method for the stable
expression of a foreign protein in a microalgae overexpression system.
To achieve the above object, the method of the present invention is
characterized in that which comprises the steps of; (i) obtaining protoplast
of
microalgae; (ii) preparing a vector containing genes coding desired proteins,
said
genes originated from organisms other than microalgae; (iii) introducing the
vector
into the protoplast to give transformed protoplast and (iv) culturing the
transformed
microalgae to produce the desired protein.
to Also, the method of the present invention could further comprise another
step of selecting transformed cells with antibiotics between step (iii) and
step (iv)
other than the above steps.
The above and other objects, features and application of the present
invention will be apparent to those of ordinary skill by the following
detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows photographs of Chlorella ellipsoidea cells stained with
calcofluor white before (a) and after (b) enzyme treatment for cell wall
removal
observed by fluorescent microscope.
Figure 2 shows a photograph of expression of GFP in transformed
Chlorella ellipsoidea observed by fluorescent microscope.
Figure 3 shows a schematic diagram of the transformation vector pCTV
Figure 4 shows the growth of transformed and non-transformed Chlorella
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ellipsoidea cultured in medium containing or not containing phleomycin.
Figure 5 shows the results of PCR amplification and Southern blot analysis
of flounder growth hormone(hereinafter fGH) gene and Sh ble gene inserted into
genomic DNA of transformed Chlorella ellipsoidea.
In figure 5, panel A shows the result of PCR amplification and Southern
blot analysis for fGH and panel B shows the result of PCR amplification and
Southern blot analysis of Sh ble; lane 1 shows molecular weight size marker
and
lane 2 shows transformed Chlorella ellipsoidea and lane 3 shows non-
transformed
Chlorella ellipsoidea and lane 4 shows fGH and Sh ble gene fragments digested
from pBluescript SK+.
Figure 6 shows the result of Western blot analysis of fGH expressed in
tranformed Chlorella ellipsoidea. In figure 6, 1 ane 1 shows molecular weight
size
marker and lane 2 shows the Glutathione-S-transferase(hereinafter, GST)-fGH
fusion protein used for production of antibody and lane 3 shows the total
protein
isolated from non-transformed Chlorella ellipsoidea and lane 4 shows the total
protein isolated from transformed Chlorella ellipsoidea.
Figure 7 shows the result of Western blot analysis showing the amount of
fGH expressed in transformed Chlorella ellipsoidea. In figure 7, lane M shows
molecular weight size marker and lane 1 & 2 show the 10~.g of GST-fGH fusion
protein and lane 3 & 4 show the fGH isolated from lOml transformed Chlorella
ellipsoidea and lane 5 & 6 show the 10~g of GST protein.
Figure 8 shows the results of Western blot analysis showing fGH
accumulated on Brachionus plicatilis and Anemia naupilus, both of which were
fed
with Chlorella ellipsoidea transformed with fGH. In figure 8, lane 1~4 show
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Brachionus plicatilis 30, 60, 90 and 120 minutes, respectively after feeding
with
transformed Chlorella ellipsoidea and lane 6~8 show Artemia naupilus 30, 60
and
90 minutes, respectively after feeding with transformed Chlorella ellipsoidea.
Figure 9 shows the growth promotion of flounder by Chlorella ellipsoidea
5 transformed with fGH. In figure 9, open bars indicate the growth promotion
by
transformed Chlorella ellipsoidea, and filled bars indicate the growth
promotion by
non-transformed Chlorella ellipsoidea, and vertical line indicates standard
deviation,
and lower cases indicate significant differences (p<0.05).
Figure 10 shows the growth promotion of flounder fries after 30 days
to feeding of Brachionus plicatilis and Artemia naupilus that had been fed for
1 hour
with Chlorella ellipsoidea transformed with fGH.
DETAILED EXPLANATION OF THE INVENTION
Throughout the specification and claims, the term of foreign protein is used
to mean any protein originated from organisms different from host microalgae,
and
it includes its active fragments, variants, and analogues as long as they
retain its
original biological activity. The term of "foreign gene" is used to indicate
any
nucleic acid sequence, regardless of its source(natural or synthetic), coding
the
foreign protein as defined above, and may include, DNA, RNA, cDNA or their
2o variants resulting form base deletion, substitution or insertion, as long
as they still
code for the foreign protein having its biological activity.
Chlorella ellipsoidea is an attractive organism for the production of complex
proteins because of its eukaryotic characteristics and low cost for large-
scale culture.
The inventors report the first functional expression of a foreign protein, the
flounder
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growth hormone(fGH) in Chlorella ellipsoidea, and the growth promotion of fish
by
feeding them this transformed chlorella. Protoplasts of Chlorella ellipsoidea
were
transformed with a vector containing the fGH gene under the control of the
cauliflower mosaic virus 35S promoter and the phleomycin resistance Sh ble
gene
under the control of the Chlamydomonas RBCS2 gene promoter. PCR
amplification and Southern blot analysis of the fGH and Sh ble genes from
chromosomal DNA isolated from the transformants confirmed stable integration
of
introduced DNA. Western blot analysis indicated that the fGH protein was
expressed in the transformed chlorella. The introduced DNA and the expressed
1o fGH were detected after seven successive transfers in media devoid of
phleomycin.
The transformed chlorella cells were first fed to zooplanktons to remove the
cellulose cell wall, and then the planktons were fed to flounder fries. These
fish
showed a 25% increase in total length and width after 30 days of feeding when
compared to control fish. These results indicate that Chlorella ellipsoidea
can be
used to produce valuable proteins at low cost.
In the present invention, the green fluorescence from chlorella transformed
with the GFP gene and the phleomycin resistance of chlorella transformed with
Sh
ble gene indicate the functional expression of these proteins. The biological
activity of the recombinant fGH was confirmed by feeding flounder fry. Thus,
in the
2o present invention, it was confirmed that the microalgae transformed with
flounder
growth hormone gene could express the hormone in a biologically active form.
Therefore, it's possible to produce valuable proteins for medicine and
industry from
transformed microalgae. In particular, microalgae could be produced with
simple
equipment and low cost, and a method of isolation and purification of
expressed
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proteins therefrom is also simple, so that the cost of producing protein could
be
significantly reduced.
Further, this invention describes the successful use of the Sh ble gene as a
selectable marker for Chlorella ellipsoidea transformation, the first
demonstration of
stable gene integration and expression of a biologically active foreign
protein in the
transformed Chlorella ellipsoidea. The results indicate that Chlorella
ellipsoidea
can be used to produce proteins of scientific or pharmacological use.
The microalgae used in the present invention are not particularly limited but
the technique can be applied to other algae including Chlorella from sea and
fresh
to water such as Chlorella ellipsoidea, Chlorella sorokiniana and Chlorella
vulgaris,
Chlamydomonas, I~olvox, Cheatoceros, Phaeodactylum, Skeletonema, Navicula,
Caloneise, Nitzschia, Thalassiosira, Amphora, Nannochloris, Nannochloropsis,
Tetraselmis, Dunaliella, Spirulina, Microcystis, Oscillatoria, Tricodesminus,
Isochryosis, Pavlova, Dinophyceae and the like.
The foreign protein gene used in the present invention is the flounder growth
hormone gene. However, other genes originated from bacteria, fungi, virus,
animals, plants or fishes could be used for overexpression by using the
present
invention.
And, vector production, cloning, transformation of host by vector, selection
2o and culture of transformant, and the recovering process of the desired
protein after
culture are known to those of skilled in the art.
The following examples are provided to illustrate the present invention,
which should not be construed to limit the scope of the present invention.
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[EXAMPLE 1] Culture and protoplast formation of Chlorella ellipsoidea
Chlorella epillsoidea was obtained from the Korea Marine Microalgae Culture
Center of Pukyong National University (Strain No. KMCC C-20). Cells were
inoculated in fresh f/2 medium(Guillard, R. R. L., and Ryther, J. H. 1962.
Studies on
marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula
confervacea(Cleve) Gran. Can. J. Microbiol. 3, 229-239) containing SO ~.g/ml
each
of chloramphenicol and streptomycin at an initial concentration of 1 X 1 O6
cells/ml and
cultured at 25°C, 18:6 hour photoperiod under a 3000 lux fluorescent
lamp. Cells
were harvested for protoplast formation 8-9 days after inoculation when the
cell
1o count reached 1-2 x108 cells/ml. Cells (SOmI) were centrifuged for Sminutes
at
1,SOOxg, washed once with 25mM phosphate buffer (pH 6.0), and suspended in Sml
of phosphate buffer containing 0.6M sorbitol, 0.6M mannitol, 4% (w/v)
cellulase(Calbiochem, USA), 2%(w/v) macerase(Calbiochem), and SO units
pectinase(Sigma Chemicals, USA). The cell suspension was incubated at
25°C for
16 hours in the dark with gentle shaking.
Protoplast formation of Chlorella ellipsoidea was confirmed in two ways. In an
osmo-stability test, the number of enzyme treated cells in distilled water
decreased
from 1.7 x 106 cells/ml to 1.0 x 105 cells/ml in 8 hours, whereas no change
occurred
in the number of untreated chlorella. This result was confirmed by calcofluor
2o white staining(Maeda, H., and Ishida, N. 1967. Specificity of binding of
hexapyranosyl polysaccharides with fluorescent brightner. J. Biochem. 62, 276-
278).
Over 80% of enzyme-treated cells were red in contrast to untreated cells that
were
blue when visualized by fluorescent microscope (see Fig. 1 ); these findings
indicated complete removal of the cellulose component of the cell wall, to
which
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calcofluor white binds.
[EXAMPLE 2] Preparation of pMinGFP and the Expression of GFP
As a first step to develop a chlorella transformation system, a small 5 kb
binary
vector was constructed from the plant transformation vector Binl9(Bevan, M.
1984.
Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12,
8711-8712). The new vector called pMIN, contains the oriV origin for
replication
in both E. coli and Agrobacterium, the npt II gene for kanamycin resistance,
the trfA
gene for DNA replication, and the right and left border T-DNA elements for
integration. The subsequent cloning of a DNA fragment containing the
cauliflower
mosaic virus 35S promoter to direct expression of the green fluorescent
protein
(GFP) produced a vector pMinGFP for use in higher plant and algae
transformation.
Chlorella protoplasts were transformed with pMinGFP by polyethylene treatment
and the expression of GFP was measured. After 7 days of culture in f/2 medium
without selection for transformation, a small number of chlorella cells
exhibited
GFP fluorescence, whereas non-transformed chlorella cells did not (see Fig.
2).
[EXAMPLE 3] Cloning the fGH gene
A flounder cDNA library was constructed with the Lambda ZAP-II cDNA synthesis
kit (Stratagene, USA) using total mRNA isolated from the Japanese flounder
pituitary gland. The titer of the amplified library was 3x109 pfu/ml and a 1~1
aliquot
2o was used for PCR amplification. The DNA fragment amplified with fGH-AN (5'-
CGGGATCCCAGCCAATCACAGA-3') and fGH-AC (5'-
CGGGCTACAGAATTC-3') primers was cloned into the pGEM-T vector (Promega,
USA) for sequence confirmation. A BamHIlNdeI fragment was subcloned into the
pGEX-3X vector (Amersham Pharmacia Biotech, USA) for glutathion-S-
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transferase-fGH (GST-fGH) fusion protein expression; this fusion protein was
used
for polyclonal antibody production.
[EXAMPLE 4] Preparation of pMinfGH
Growth hormone genes have been cloned from several fish species and their
growth-
5 enhancing effects have been observed in transgenic fish. The growth hormone
gene (fGH) from the Japanese flounder, Paralichthys olivaceus, the major
aquaculture fish in Korea, was used to transform chlorella. The fGH gene was
cloned by PCR amplification of a flounder pituitary cDNA library, using the
fGH-N
primer (5'-CGGGATCCGGTCAGTCCCTTATGCAGCCAATCACA-3') and fGH-
1o C primer (5'-AAAAGCTCGAGCTCTTGGCGGAG-3') (Watahiki, M., Yamamoto,
M., Yamakawa, M., Tanaka, M. & Nakashima, K. 1989. Conserved and uniques
amino acid residues in the domains of the growth hormone: flounder growth
hormone deduced from the cDNA sequence has the minimal size in the growth
hormone prolactin gene family. J. Biol. Chem. 264, 312-316). Replacement of
the
GFP gene in pMinGFP vector by the 560 by PCR product resulted in vector
pMinfGH.
[EXAMPLE 5] Preperation of pCTV
We used the Sh ble gene, originated from Streptoalloteichus hindustamus, which
encodes a small protein (13.7kDa) that confers resistance to tallysomycin,
2o bleomycin, phleomycin, and zeomycin by binding to the antibiotics and
inhibiting
their DNA cleaving activities. To determine if Chlorella ellipsoidea was
inhibited
by phleomycin, the alga was cultured in f/2 medium containing different
concentrations of phleomycin; Reduced growth occurred in media containing 0.1
or
0.5 ~ g/ml phleomycin, and the alga failed to grow in media containing more
than 1
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~glml phleomycin. Thus, the Sh ble gene that confers resistance to phleomycin
is
suitable to select transformed chlorella The Sh ble coding region and upstream
Chlamydomonas reinhardtii RBCS2 promoter were amplified from the ~plasmid
pSP109(Lumbreras, V, Stevens, D. R., & Purton, S. 1998. Efficient foreign gene
expression in Chlamydomonas reinhardtii mediated by an endogenous intron.
Plant J.
14, 441-447) with ble-N primer (5'-AAACTCGAGGGCGCGCCAGAAGGAGC-
3') and ble-C primer (5'-AAACTCGAGAATTCGAGGTCGGTACC-3'). The 880
by PCR product was digested with Xho I and subcloned into pMinfGH to construct
the chlorella transformation vector pCTV (see Fig. 3).
[EXAMPLE 6] Transformation of Chlorella ellipsoidea with pCTV vector
Chlorella protoplasts( 1 x 1 Og) were centrifuged at 400xg for 5 minutes,
resuspended in
5 ml of f/2 medium containing 0.6M sorbitol/mannitol, centrifuged at 400Xg for
S
minutes, and resuspended in lml of 0.6 M sorbitol/mannitol solution containing
O.OSM CaCl2. Then, 1 X 108 protoplasts in 0.4m1 were placed in a fresh
microcentrifuge tube and S~g of pCTV vector was added with 25~g calf thymus
DNA(Sigma Chemicals). After 15 minutes incubation at room temperature, 2001
of PNC[0.8 M NaCI, 0.05 M CaCl2, 40 % PEG 4000(Sigma Chemicals)] was added
and mixed gently for 30 minutes at room temperature. Then, 0.6 ml f/2 medium
supplemented with 0.6 M sorbitol/mannitol, 1 % yeast extract and 1 % glucose
was
2o added, and the cells were incubated at 25°C for 12 hours in the dark
for cell wall
regeneration. The cells were transferred to fresh f/2 medium containing
phleomycin (l~g/ml) and cultured as described above.
Detectable growth occurred by 5 days and the cell growth reached stationary
phase
by 15 days. In contrast, no detectable growth occurred in non-transformed
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protoplasts(see Fig. 4). The slow growth of the transformed chlorella cells
was
consistent with the preliminary transformation experiments with pMinGFP, where
only a small percentage (2%) of the cells displayed green fluorescence. When
the
transformed chlorella cells in the stationary phase were transferred to fresh
f/2
medium or f/2 medium containing phleomycin, no detectable growth differences
occurred. Furthermore, the growth rates in the two media were similar to the
growth of non-transformed chlorella in f/2 medium lacking phleomycin (see Fig.
4).
These results indicate that the introduced DNA has no effect on chlorella
growth;
also the transformed cells did not exhibit any morphological changes.
to [EXAMPLE 7] Stable integration of introduced DNA
Stable integration of introduced DNA into chromosomal DNA is a prerequisite
for
use of chlorella as an expression system. PCR and Southern analyses were
performed to determine if the introduced DNA was integrated into the chlorella
chromosomal DNA.
(Al. DNA isolation.
Approximately 3x108 transformed cells were pelleted from 3 ml of culture,
resuspended in 500 ~l of CTAB buffer[250m1: hexadecyltrimethylammonium
bromide(CTAB) Sg, 1M Tris(pH 8.0) 25m1, NaCI 20.45g, EDTA 1.68g, (3-
mercaptoethanol(2%)] and incubated at 65°C for 1 hour, and then
extracted with an
2o equal volume of phenol/chloroform. The aqueous phase recovered after 5
minutes
centrifugation at 3,OOOxg was extracted several times and chromosomal DNA was
precipitated with. ethanol, pelleted and resuspended in 30 ~1 of TE buffer.
(B). PCR and Southern blot analysis.
fGH-N/fGH-C and ble-N/ble-C primer pairs were used to amplify the fGH gene and
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the Sh ble gene from isolated chromosomal DNA, respectively. 200ng of
chromosomal DNA and 100pmole of each of the primers were added to 50 p1
reactants, and subjected to 30 cycles of 1 minute denaturation at 94°C,
30 seconds
annealing at 54 or 57°C for the fGH and the Sh ble genes, respectively,
1 minute
extension at 72°C followed by 5 minutes extension at 72°C.
Probes for Southern
blot were synthesized using the DIG-DNA labeling kit (Boehringer Mannheim,
Germany).
PCR products of the expected size were produced only with DNA isolated from
transformed chlorella. These DNA fragments were identified by Southern
analyses
to with probes specific to the fGH or Sh ble genes(see Fig. 5). The stability
of the
integrated DNA was confirmed by PCR amplification of the two genes from the
chromosomal DNA isolated from chlorella after seven serial transfers into
medium
lacking phleomycin.
[EXAMPLE 8] Expression of fGH in tranformed Chlorella ellipsoidea
fGH expression was tested by Western analysis as described hereinafter.
Transformed Chlorella ellipsoidea was harvested from 3 ml of culture
containing
10$ to 109 cells by centrifuging for 5 minutes at 17,000 x g. The cells were
homogenized in liquid nitrogen, resuspended in 20 p1 of sample loading
buffer[1mM
EDTA, 250mM Tris-Cl (pH 6.8), 4 % SDS, 2 % ~' - mercaptoethanol, 0.2
2o bromophenyl blue, 50 % glycerol], and boiled for 10 minutes. The sample was
centrifuged for 10 minutes at 12,000 x g and the supernatant was
electrophoresed on
a 15 % SDS-PAGE. Also, protein extracts prepared from non-transformed
chlorella
were separated by SDS-PAGE. Western blot analysis was conducted by standard
procedures. Protein extracts separated from tramsformed and non-transformed
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chlorella by SDS-PAGE were transferred onto nitrocellulose membranes. The
final
dilution of polyclonal antibody against fGH was 1:3,000 and alkaline
phosphatase-
conjugated anti-mouse IgG was used as the secondary antibody.
The 20kDa fGH was present in transformed chlorella but absent in non-
transformed
s cells (see Fig. 6).
One requirement for a successful expression system is that the foreign protein
be
produced at high level. The amount of fGH expressed in transformed chlorella
was
determined by an Enzyme Linked Immunosorbent Assay (ELISA) and Western blots
with purified GST, GST-fGH fusion protein and extract from transformed
chlorella
to using polyclonal antibody against GST-fGH fusion protein(see Fig. 7). About
400ng of fGH was obtained from 1 X 10g stationary phase cells (400p g of total
protein
in lml culture). The yield is equivalent to 400~g fGH per litter of cultured
chlorella
assuming a final cell count of 1x10$ cells /ml. Considering the low cost of
culture
medium for the alga, this system could be used to produce eukaryotic proteins,
15 especially proteins of pharmaceutical importance.
[EXAMPLE 8] Biological activity test
Although chlorella can not be directly fed to fish and crustacean larvae
because of
the high cellulose content in their cell walls, chlorella have been used to
mass
culture zooplanktons, which contain cellulase. Also it is known that fish can
take
2o up proteins in feed by pinocytosis and there are reports of fish growth
promotion by
the oral administration of recombinant mammalian and fish growth hormone.
Thus, four day old flounder larvae were grouped into 1000 fish each in a 300
litter
tank filled with 200 litter of sea water. Rotifers (Brachionus plicatilis) and
brine
shrimp (Anemia nauplius) were used to accumulate the growth hormone and to
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remove the cellulose from chlorella cell wall. Zooplanktons were starved for
one
day after hatching and provided with 3x108 cells/ml of transformed and non-
transformed chlorella for one hour. Western analysis confirmed that the fGH in
the
alga accumulated in zooplankton bodies by 1 hour of feeding; after 1 hour fGH
was
5 degraded and disappeared 2 hours after feeding (see Fig. 8). The flounder
larvae
were fed once a day with the rotifers for 10 days and then with a mixture of
the
rotifers and the brine shrimp for 5 days, followed by 15 days feeding with the
brine
shrimp. The final counts of the rotifer and the brine shrimp were 10 and 5
individuals/ml, respectively. Four day old flounder fries were cultured for 30
days
10 with zooplanktons enriched for 1 hour with transformed and non-transformed
chlorella. The lengths of the fish larvae were measured after 10 day feeding
and both
the length and width of the larvae were measured after 30 day feeding. Fifty
randomly selected fish were measured from each of three replicates containing
1000
fish. As shown in Fig. 9, the length of the fish differed significantly after
10 days
15 and had a 25% increase in both length and after 30 days (see Fig 10).
Although preferred embodiments of the present invention have been
described in detail herein above, it should be clearly understood that many
variations
and/or modifications of the basic inventive concepts herein taught which may
appear
2o to those skilled in the art will still fall with in the spirit and scope of
the present
invention identified in the appended claims.
