Construction of a Shuttle Vector for Heterologous Gene Expression in Escherichia coli and Microalgae Anabaena

doi:10.4236/eng.2013.510B111

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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

Email: *[email protected]

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

Forward

5′-CCGGAATTCACGCCATAATC

ATGTGTC-3′ EcoRI

Reverse

5′-TGCACTGCAGTGGGGATAAT

TACAACTC-3′

PstI

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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