Engineering, 2013, 5, 540-544
http://dx.doi.org/10.4236/eng.2013.510B111 Published Online October 2013 (http://www.scirp.org/journal/eng)
Construction of a Shuttle Vector for Heterologous Gene
Expression in Escherichia coli and Microalgae Anabaena
Donghui Song*, Jing Li, Xiaoxu Hu, Bo Xi
Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science & Engineering,
Tianjin University of Science &Technology, Tianjin, China
Received 2013
ABSTRACT
The construction of an integrative shuttle expression v ecto r and potential utility was reported in Escherichia coli and
Anabaena (Nostoc) sp. strain PCC 7120. The vector comprised of the following elements: (a) an intergenic non-coding
region from Anabaena to facilitate its genomic integration (b) a strong functional PpsbAI promoter from Anabaena for
desired gene expression and (c) neomycin phosphotransferase gene with its own promoter for the selection of transfor-
mants. The constructed vector pAnFP was evaluated by cloning, transfer and expression of the gfp gene encoding green
fluorescent protein. When the E. coli and Anabaena sp. strain PCC 7120 were transformed, intensive green fluorescence
produced by the products of GFP protein was observed. This resul t indicated that the integrative shuttle vector pAnFP
can be promisingly used in genome transformation for ex pression of heterologous genes in E. coli and microalgae such
as Anabaena and Nostoc strains.
Keywords: Anabaena sp. PCC 7120; Integrative Shuttle Vector; pAnFP; gfp Gene
1. Introduction
In the last few years, microalgae have been extensively
investigated for biotechnological applications as a rich
source of bioactive compounds [1], for biofuels produc-
tion [2,3], and for nitrogen biofertilizer [4]. Filamentous
nitrogen fixing microalgae Anabaena have been particu-
larly interesting since they harbor vital processes of pho-
tosynthesis and nitrogen fixation [5,6]. Apart from much
research on heterocyst development, Anabaena strains
have been known for their ability to overproduce bio-
hydrogen for potential utilization as a clean and renewa-
ble biofuels [7,8]. Genetic engineering is one approach
for microalgal strains to produce more lipids-rich cells.
Two primary requirements must be fulfilled before a
microalgal strain can be successfully engineered in this
manner. One is that these expressible and recombinant
genes which affect lipid metabolism must be available;
another requirement is often effective approach which
should be developed to incorporate stably cloned ex-
ogenous genes into host cells [9]. Strain Anabaena (Nos-
toc) sp. PCC 7120 (referred to in this paper as Anabaena
7120) has been chosen for this study since its genome is
completely sequenced and it is amenable to genetic ma-
nipulation. Mor e importantly is that the development of
stable integrative express ion system will benefit from the
well developed molecular genetics of Anabaena 7120.
Therefore, we reported here a construction of integrative
shuttle vectors, pAnFP, for inserting exogenous genes
into microalgae Anabaena 7120.
2. Materials and Methods
2.1. Strains and Culture Conditions
Bacterial strains and plasmids used in this study are
shown in Table 1. Anabaena 7120 was grown in BG11
medium without nitrate [10], pH 7.2, at 27˚C. E. coli
strains were grown in Luria Bertani (LB) medium with
appropriate antibiotics when required. Antibiotics were
used at 100 μg/ml Ampicillin (Amp) and 50 μg/ml ka-
namycin (Km) for E. coli and 25 μg/ml neomycin (Nm)
in BG11 agar me dia or 12.5 μg/ml in liquid BG11 media
for Anabaena transformants.
2.2. Molecular Biology Methods
Standard molecular biology techniques were used [11].
Anabaena 7120 chromosomal DNA was isolated as de-
scribed [12]. DNA fragments from Anabaena 7120 were
amplified using th e polymerase chain reaction (PCR)
with appropriate primers shown in Table 2. The nptII
gene (encoding neomycin phosphotransferase) and gfp
gene (encoding green fluorescent protein) was indepen-
dently PCR amplified from plasmid pET-30a(+) and
*Corresponding author.

D. H. SONG ET AL.
541
Table 1. Bacterial strains and plasmids.
Strains and plasmids Characteristics Source
Bacterial strains
E. coli TOP10 recA∆lacU169 Invitrogen
Anabaena (Nostoc) sp.
strain PCC7120
wild type Pasteur
Plasmids
pET-30a(+) Kmr Novagen
pGFP Amp r Clontech
pBluescript II SK (+) Amp r TakaRa
pTF2 A mpr This study
pTF1 A mpr This study
pTPpsbAI-F2 Amp r This study
pTF1-PpsbAI-F2 Amp r This study
pAnFP Amp r ,Kmr This study
pAnFP-gfp Amp r ,Kmr This study
pGFP, respectively. Re-introduction of plasmids harbor-
ing with gene gfp by triparental conjugation [13] into
Anabaena 7120 wild-type cells were used to determine
whether these plasmids were responsible for the gene
expression and growth phenotypes. Complete nuc leotide
sequence analysis of all the components of pAnFP vector
was carried out by dideoxy sequencing method using
appropriate primers (Table 2). The sequence identity of
cloned fragments with the known Anabaena 7120 ge-
nome sequence was evaluated using the BLAST algo-
rithm (GenBank database, http://blast.ncbi.nlm.nih.gov/,
and Kazusa DNA Research Institute, http://genome.ka-
zusa.or.jp/cyanobase/Anabaena).
2.3. Fluorescence Microsc opy
The green fluorescence expressing images of E. coli and
Anabaena 7120 were obtained by light excitation at 365
nm and were captured by light emission at 510 nm with
an AxioCam HRc camera attached to a Carl Zeiss LSM
510 META NLO microscope.
3. Results
3.1. Construction of an Integrative Shuttle
Expression Vector pAnFP
The plasmid pBluescript II SK (+), hereafter referred as
pBS-T, was chosen for construction of the integrative
expression shuttle vector pAnFP comprised of (1) flank-
ing regions F1 and F2 for integration, (2) Anabaena
promoter (PpsbAI) for expression of the downstream gene,
(3) the nptII ge ne for selection of the transformants with
Table 2. Primers designed for pcr amplification.
Components Sequences Restriction
sites
F1
Forward
5′-CCGGAATTCACGCCATAATC
ATGTGTC-3′ EcoRI
Reverse
5′-TGCACTGCAGTGGGGATAAT
TACAACTC-3′
PstI
F2
Forward
5′-CGCGGATCCCCCCAATTTTG
AAACCTATC-3′ BamHI
Reverse
5′-CTAGTCTAG AGGCTCACCATA
GGTGAATGCAC-3′
XbaI
PpsbA1
Forward
5′-GAGCTGCAGGGATTCCCAAA
GATAGGG-3′
PstI
Reverse
5′-CTCGGATCCCCATATGTTTTT
ATGATTGCTTTG-3′
BamHI/
NdeI
nptII
Forward
5′-CGGGATCCCATCGATACTCA
CGTTAAGGGAT-3′
BamHI/
ClaI
Reverse
5′-CGGGATCCCAGGTGGCACTTT
TCGGGGA-3′ BamHI
gfp
Forward
5′-CCGCATATGACATATGAGTAA
AGGAGAACAAC-3′
NdeI
Reverse
5′-CGCATCGATTTATTTGTATAGT
TCATCC-3′ ClaI
Underlined and bold regions of the primer sequences represent the incorpo-
rated restriction endonuclease sites.
its own promoter in transgenic Anbeana, and (4) ampicil-
lin marker on pBS-T for the positive selection of recom-
binants in E. coli. The 931 bp F region (for flanking) was
selected from an 1192 bp intergenic, non-coding region
separating the ORFs alr3857 and alr3858 of Anabaena
7120 genome (Anabaena chromosome 4654700-4655631).
Individual elements of the pAnFP vector were PCR am-
plified using specific primer pairs shown in TABLE II
and sequentially cloned at indicated sites (Figure 1(a)).
The F region was amplified separately as two fragments
F1 and F2. The vector pAnFP was constructed in three
steps (Figu re 1(b)): (a) The 431 bp F2 fragment was
PCR amplified from Anabaena 7120 DNA, restriction
digested with the enzymes BamHI and XbaI and ligated
to pBS vector at BamHI and XbaI sites. A putative psbAI
promoter region was selected from the upstream region
of the psbAI gene, which encodes the D1 protein of pho-
tosynthetic apparatus. A 182 bp PCR amplified PpsbA I
product was restriction digested with the enzymes PstI
and BamHI and ligated to construct pTF2 at identical
sites. The resulting construct pTPpsbAI-F2 on indepen-
dently dige stion w i th Xb aI/BamHI and X b a I/PstI re-
leased the 431 bp F2 fragment and F2 + PpsbAI fragment
(Figures 2(a) and (b)). An NdeI site introduced in the
PpsbAI reverse primer (TABLE II) provided an appropriate

D. H. SONG ET AL.
542
(a)
(b)
Figure 1. Construction of integrative shuttle vector pAnFP. (a) Schematic diagram of the integrative expression cassette,
containing two flanking regions F1 and F2 for homologous recombination, a promoter PpsbAI, appropriate cloning sites
downstream to the promoter (NdeI-ClaI) and a nptII gene, cloned in pBS-T. (b) Flow diagram of pAnFP construction
showing the precursor vector along with steps (a), (b), and (c).

D. H. SONG ET AL.
543
(a) (b) (c) (d) (e)
Figure 2. Experimental verification of pAnFP construction. (a) The construction of clone pTPpsbA1-F2, containing PpsbA1
promoter and F2 cloned into pBS. M: DNA Marker III; lane 1: pTPpsbA1-F2 digested with XbaI/BamHI. (b) The con-
struction of clone pTPpsbA1-F2, containig PpsbA1 promoter and F2 cloned into pBS. M: DNA Marker III; lane 1: construct
pTPpsbA1-F2 digested with PstI/ XbaI. (c) The construction of pTF1-PpsbA1-F2. M: DNA D2000 plus; lane 1: pTF1-PpsbA1-F2
digested with EcoRI/PstI; lane 2: pTF1-PpsbA1-F2 digested with XbaI/BamHI; lane 3: pTF1-PpsbA1-F2 digested with
PstI/BamHI. (d) The final assembly of pAnFP vector, containing F1, F2, PpsbA1 promoter and nptII gene. M: DNA D2000 plus;
lane 1: pAnFP digested with EcoRI/PstI; lane 2: pAnFP digested with XbaI/BamHI; l ane 3: pAnFP digested with PstI/
BamHI; lane 4: pAnFP dige- sted with BamHI. (e) Identification of pAnFP-gfp digestion with NdeI/ClaI. M: DNA D2000 plus.
site for the cloning and expression of a desired gene in
the vector. (b) A 500 bp PCR amplified F1 fragment was
digested with EcoRI and PstI, ligated to pBS vector at the
same sites, and designated as construct pTF1. The 613 bp
XbaI-PstI fragment carrying PpsbAI + F2 from construct
pTPpsbAI-F2 was ligated into construct pTF1 at the XbaI
and PstI sites. The resulting construct pTF1-PpsbAI-F2 on
digestion with XbaI and PstI released the 500 bp F1
fragment (Figure 2(c), lane 1). (c) Neomycin phosphor-
transferase gene (nptII), conferring resistance to neomy-
cin, was amplified from the plasmid pET-30a(+), using
nptII forward and reverse primers (TABLE II) as a 985
bp PCR product. This fragment was digested with Bam-
HI to obtain a 985 bp fragment which was ligated into
the same sites of pTF1-PpsbAI-F2, resulting in the con-
struction of a 5.082 kb integrative expression vector,
pAnFP. The vector pAnFP on digestion with the en-
zymes released 500bp F1, 431bp F2, 182bp PpsbAI and
985bp nptII (Figure 2(d)).
3.2. Expression of the Shuttle Vector pAnFP
with Gfp Gene in Escherichia coli and
Anabaena 7120
Green Fluorescent Protein (GFP) has been used as a
marker of gene expression in microalgae [14]. To eva-
luate ability of pAnFP as a po tential integrative shuttle
expression vector, a gfp gene from the pGFP plasmid
was PCR amplified by using gfp primers (Table 2). The
717 bp PCR amplified DNA fragment was restriction
digested with NdeI and ClaI and cloned into the NdeI
and ClaI digested pAnFP vector to yield plasmid
pAnFP-gfp. Digestion of pAnFP-gfp with NdeI and ClaI
yielded 717 bp (gfp) and 5 kb pAnFP fragments (Figure
2(e)). The inserted gene was transformed into E. coli
TOP10 and Anabaena 7120 as described in methods,
respectively. Transformants of E. coli and Anabaena
7120 harboring with the recombinant vector pAnFP-gfp
were observed strong green fluorescent expression (Fig-
ure 3)). This result showed that the integrative vector
pAnFP-gfp has successfully expressed in both E. coli and
Anabaena 7120.
4. Discussion
The expression of gfp gene in E. coli and Anabeana 7120
indicated that th e integr ativ e sh u ttle expres sion vector
pAnFP can be promisingly used in genome transforma-
tion of cyanobacteria such as Anabaena strains. Plasmid
pAnFP contains an integrative cassette and offers a novel
combination of many desirable features. A gene of inter-
est can be cloned downstream of a strong PpsbAI promoter
of Anabaena, integrated in the chromosome of Anabaena
at an innocuous location and expressed. The vector
pAnFP makes it more attractive for application s in the
chromosomal integration and makes it amenable for
subsequent tracking. Moreover, using of a promoter dri-
ven by a naturally available stimulus is eco-friendly and
does not change the circadian expression of genes of mi-
croalgae [15]. The PpsbA1 promoter from other sources has
earlier been us ed for expressi o n of desired genes in
Anabaena [16] and in other cyanobacteria [17] and plants
[18]. Our findings indicate that the pAnFP vector bearing
heterologous genes can be successfully expressed in E.
coli and be used for transformation of cyanobacteria such
as Anabaena or Nostoc strains in the future. The integra-
tive shuttle expression vector pAnFP may prove the way
to generate desired transgenic microalgae for the bioactive

D. H. SONG ET AL.
544
Figure 3. Expression of gfp gene in E. coli and Anabaena
7120. Cells were excited at 365 nm and emitted at 510 nm to
visualize green fluorescence of GFP. (A) E. coli strains car-
rying pAnFP-gfp. (B) Anabaena 7120 strains carrying
pAnFP-gfp.
compounds or biofuels production which can be varying
potential utility of basic research and industrial applica-
tions.
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