Synthetic Gene Paper

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JOURNAL of BIOTECHNOLOGY RESEARCH in TROPICAL REGION, Vol. 1, Oct. 2008 (Special Edition) ISSN: 1979-9756

Construction of a CSF3-Synthetic Gene for Recombinant Human G-CSF Expression in Yeast Using a TBIO

(Thermodynamically Balanced Inside-Out) Method 1

Construction of a CSF3-Synthetic Gene for RecombinantHuman G-CSF Expression in Yeast Using a TBIO(Thermodynamically Balanced Inside-Out) Method

Asrul Muhammad Fuad1*, Dian Fitria Agustiyanti1, Yuliawati1, Citra Fidyani1,Aminah1, Adi Santoso1

1Bioprocess Engineering Laboratory, Bioprocess Division, Research Center for Biotechnology,LIPI. Cibinong Science Center-Jalan Raya Bogor Km. 46, Cibinong 16911-Bogor

AbstractHuman Granulocyte-colony stimulating factor (hG-CSF or G-CSF) is a cytokine, which has therapeutic

applications. It is a hematopoietic growth factor thatstimulates proliferation of granulocytic cells calledgranulopoiesis. It is used to increase neutrophilicgranulocytes or neutrophils level in the body. Neu-trophils compose the majority of white blood cell(WBC) components and play very important roles inhuman defense against infections. Neutropenia isan abnormal condition where there is very low num-ber of WBCs in human body due to various causessuch as drug side-effects, vitamin B12 defi-ciency,cancer chemotheraphy, virus infections and bonemarrow cells abnormality. Nowdays, neutronnpeniacould be avoided by the administration of recombi-

nant hG-CSF preparation. G-CSF is a monomerprotein encoded by a single gene called CSF3.There are two variants of G-CSF found in the body.However, both variants (177aa and 174aa) showsimilar bioactivity. Recombinant hG-CSF has beenused widely in combination with various anticancerdrugs to fight cancers. It was used to avoid neutro-penia and complication during and after chemothe-rapy. This research had a goal to produce recombi-nant(s) hG-CSF and its analogs (muteins) with im-proved characteristic(s) using synthetic gene(s) thatcode for this protein. The recombinant protein will beexpressed in a defined yeast expression system. To

achieve this goal, we firstly had to construct aCSF3-synthetic gene containing optimized codon forexpression in Pichia pastoris. At present, we suc-cessfully constructed a version of CSF3-syntheticgene using a PCR-based technique called the TBIO(Thermodynamically Balanced Inside-Out) method.

*Correspondence to:Dr. Asrul Muhammad Fuad

Tel: +62 21 8754587; Fax: +62 21 8754588e-mail: [email protected]

A DNA sequence of 558bp long has been con-structed using 14 oligonucleotides with an averagelength of 60 nucleotides. The synthetic gene se-

quence and oligonucleotides used had been de-signed to contain yeasts codon preferences with theDNAWorks3.1 program. It was cloned in a commer-cial cloning vector and is undergoing DNA sequenceanalyses.Keywords: recombinant hG-CSF, CSF3, neutrope-nia, neutrophils, granulopoiesis, synthetic gene,TBIO, Pichia pastoris.

INTRODUCTION

G-CSF (granulocyte-colony stimulating factor) is

a haematopoietic growth factor that works by en-

couraging the bone marrow to produce more white

blood cells. Growth factors are special proteins pro-

duced naturally in the body. They can also be made

as drugs. One of the main side effects of chemothe-

rapy drugs is the reduction in the number of white

blood cells. This makes our body less able to fight

infection. There is a risk that one could develop a

serious infection, which might have to be treated in

hospital. If the number of blood cells (blood count)

is low when the next dose of chemotherapy is due,

then the chemotherapy may have to be postponed orthe dose lowered.

In this situation, G-CSF can be given to stimulate

the bone marrow to produce new white cells more

quickly after chemotherapy. This can shorten the

period during which the patient is at risk of develop-

ing a serious infection. G-CSF is not needed with all

types of chemotherapy treatment, as the white blood

cell count can often recover on its own. G-CSF may

sometimes be used before high-dose chemotherapy

to make the bone marrow produce more stem cells.

These extra stem cells can then be collected and

given back to you after high-dose chemotherapytreatment. The stem cells then go back into the bone

marrow and produce blood cells.


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Fuad, et.al. 2

Human G-CSF was coded from a single gene

called CSF3. It was mapped on the chromosome 17.

There are two isoforms of mature protein G-CSF

mostly found in the body, which are 177 aa

(NP_000750) and 174 aa (NP_757373) in lengths.

Both isoforms show similar bioactivities. There are

three different types of G-CSF commercially availa-

ble; lenograstim (Granocyte), filgrastim (Neupo-

gen) and pegylated filgrastim (Neulasta). These

drugs work in a similar way. The molecules of the

pegylated filgrastim have more glycosylation that

helps the drug to work longer.

Chemical synthesis of DNA sequences provides

a powerful tool for creating, modifying and studying

gene function; such as studying its structure and

expression in a given host cell. In the past, the most

direct method to construct a synthetic gene were to

mix overlapping preformed double stranded DNAand ligate them each other enzymatically. However,

the yield of the full-length product declines sharply

with increasing number of DNA duplexes. The more

common method is to construct separate DNA seg-

ments of the gene from a smaller number of DNA

duplex, amplify each fragment by sub-cloning into a

plasmid vector then ligate the fragments to give the

full-length gene. Although, this method could effi-

ciently produce the intermediate product for each

step, the intermittent sub-cloning and bacterial am-

plification steps make the procedure very tedious

and time-consuming. The more recent method in-volves assembling of several synthetic primers or

oligonucleotides to produce up to 500 bp DNA se-

quence, assembled in a single-tube annealing and

ligation reactions.

In this study, we applied a method for a synthet-

ic gene construction called the TBIO (Thermody-

namically Balanced Inside-Out) method. This me-

thod, originally reported by Gao et al. (2003), offers

a very efficient method for gene construction with-

out the use of restriction and ligation procedures.

This method is a PCR-based single-step DNA syn-

thesis uses both primers in sense and anti-sensestrands each for half of the gene length. The primer

elongation will run on both directions. Thus, TBIO

bidirectional elongation must be completed for a

given outside primer pair before the next round of

bidirectional elongation can take place. The method

was reported to be successfully used in constructing

some DNA sequences of up to 1712 bp in length.

The same method was used to generate longer DNA

sequences in a slightly modified method (Xiong et

al., 2004). The method was reported to give high-

fidelity and cost-effective PCR-based two-step

DNA synthesis for construction of long segments of

DNA. The long DNA sequence (2,382 bp) was dis-

sected into five DNA segments of around 500 bp in

length. Each of which was constructed with the

TBIO method.

The purpose of this study was to synthesize a

yeast-codon-optimized CSF3 gene which might be

expressed extracellularly from a methylotrophic

yeast Pichia pastoris. An mRNA variant of human

CSF3 gene (variant-2) producing the hG-CSF mole-

cule isoform-b, was altered to obtain a CSF3 syn-

thetic gene with optimized-codon preference for

protein expression in P. pastoris. In this research,

we constructed a DNA sequence or an ORF (Open

Reading Frame) of hG-CSF gene, called CSF3, with

the TBIO method. The method was slightly mod-

ified in which the PCR reactions was run sequential-

ly with a given primer pair from the middle of the

gene. The PCR product was then used as template

for sequence elongation with the next primer pair of

the outside part of the sequence being generated.The amino acid sequence of the hG-CSF iso-

form-b (NP757373) was used as template for gener-

ation of the synthetic gene sequence. The signal

peptide (first 30 amino acid) was excluded from the

sequence, resulting in a protein sequence of 174 aa

in length.

MATERIALS AND METHODS

Materials: The oligonucleotide primers were syn-

thesized by Generay Biotech. The high fidelity PfuDNA polymerase and dNTPs mix were purchased

from Fermentas. Cloning plasmid InstanCloneTM

was purchased from Fermentas. Plasmid DNA gel

extraction kit was from RBC. The XhoI and SalI

restriction enzymes were from Fermentas. T4 DNA

ligase was from Fermentas.

CSF3 Open Reading Frame (ORF) and primerdesign: The CSF3Open Reading Frame (ORF) was

generated based on the protein sequence of the iso-

form-b of hG-CSF molecule having 174 amino acid

residues. The protein sequence was retrieved fromthe gene database (GenBank accession no.

NP_757373) or the protein database (SwissProt ac-

cession no. P09919-2). The synthetic gene excludes

the first 30 aa native signal peptide. The DNA frag-

ment of the synthesized CSF3 ORF was 522 bp in

length. However, in the design of the CSF3 ORF,

two restriction sequences (XhoIand SalI) were add-

ed at both ends of the synthetic gene as well as a

linker peptide (KREAEA) at the 5 end. The result-ing ORF sequence was 558 bp in length. The protein

sequence was submitted to a software program,

DNAWorks 3.1, by which the oligonucleotide se-

quence (ORF) of the synthetic gene was then gener-

ated. The gene was optimized to contain P. pastoris

codon preference. Some parameters was set up for


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the required oligonucleotide synthesis, including

primer length (set at 60 nt), annealing temperature

(set at 60oC), codon frequency threshold (set at

10%) and the output mode was set to TBIO method

(thermodynamically balanced inside-out). The pro-

gram sent a detail output of the DNA sequence as

well as primer sequences required for construction

of the synthetic gene. There were 14 primers or oli-

gonucleotides which should be synthesized, each

having 60 nt (nucleotides) in average and an overlap

region varied from 18 to 25 nt between adjacent

primers.

CSF3Open Reading Frame construction and ex-perimental design: The synthetic gene was con-

structed according to a modified TBIO method (Gao

et al., 2003). The PCR-based primer extention me-

thod of the synthetic gene, which is the basic prin-ciple of the TBIO method, was started from the

middle of the gene sequence. As shown in Fig. 1,

the 3-terminal ends of the first pair of 60 mersense- and antisense-strand TBIO primers (P7 and

P8) overlap in the middle of the synthetic gene se-

quence. The gene synthesis started at this point by

primer extension process. The PCR reaction was

continued with the next 60 mer pair of outer primers

(P6 and P9), both from sense- and antisense-strands.

The reaction was repeated with the next pair of pri-

mers. Those pairs of primers were added sequential-

ly to extend the sequence polymerization in bothdirections ofsense and antisense strands. The PCR

mix reaction uses 40 nM of each primer pairs, 0.2

mM dNTPs, 1x Pfu buffer and 1.25 U ofPfu DNA

polymerase in a 50 l PCR mix reaction volume.

For the subsequent PCR reaction, 2.5 l of the inner

DNA sequence was added (as template) into the

next PCR reaction with the next pair of primer for

DNA sequence elongation. The PCR cycles used

was 2 min for first denaturation at 95oC, for 1 min

of denaturation at 95oC, for 30 sec of annealing at

59oC, for 1 min of elongation at 72

oC and 5 min for

final elongation at 72oC. The PCR reaction was setfor 25 cycles.

Cloning and analysis of the synthetic gene: The

PCR products of each sequential PCR reactions

were analyzed in a 1.5% agarose gel electrophoresis.

The final length of the synthetic DNA sequence be-

ing constructed is 558 bp. Into this PCR product, an

amount of 1 to 2 U of Taq DNA polymerase (Fer-

mentas) was added and the mixture was incubated at

72oC for at least 30 min. This process was carried

out in order to add an additional A (adenine) atthe 3-end of each double stranded synthetic DNAsequence which has been produced. The DNA prod-

uct was then sub-cloned into a commercial A/T

cloning plasmid kit such as InstanCloneTM

pTZ57R/T (Fermentas). The recombinant plasmid

was transformed into E. coli XL1-Blue. The recom-

binant clones were then selected on selection LB-

agar medium containing IPTG and X-gal. Positive

clones (white colonies) were selected, cultured in an

appropriate media and the recombinant plasmids

were extracted and analyzed. Enzymatic restriction

analysis was then carried out to analyze the recom-

binant plasmids using single and double-digestion

analysis (with XhoI and SalI). Cloning and trans-

formation into the cloning plasmid kit were carried

out according to protocols given by the producer.

Plasmid preparation and restriction analysis were

done using the general protocols for molecular clon-

ing according to Ausubel et al. (2002). The recom-

binant plasmid(s) harboring the correct DNA insert

was then submitted for DNA sequence analysis us-

ing appropriate primers.

RESULTS AND DISCUSSION

CSF3 Open Reading Frame (ORF) and primerdesign: Human G-CSF is encoded by a single gene

called CSF3 belongs to IL-6 superfamily. The gene

is located in chromosome-17 and mapped at locus

17q11.2-q21 by in situ hybridization (Tweardy et

al., 1987). The gene produces 3 variants of mRNA

that resulted in 3 different types of preprotein hG-

CSF. However, there are two isoforms of maturehG-CSF mostly found in the body; isoform-a (177

aa, GenBank accession no. NP-000750) and iso-

form-b (174 aa, GenBank accession no. NP-

757373). Although isoform-b lacks three amino ac-

ids VSE at position 66-68, both isoforms show simi-

lar bioactivity.

In this study, the protein sequence of the short ver-

sion of hG-CSF (isoform-b) was used as template to

generate the synthetic CSF3 gene sequence

(CSF3syn). The human CSF3 gene that codes for

hG-CSF was codon-optimized for expression in

yeast. However, the first 30 aa native peptide signalwas excluded in the synthetic gene design. Instead, a

linker peptide KREAEA was added at the N-

terminal of the sequence. The linker peptide

presents proteolytic cleavage site(s) that will be use-

ful for secretion of the recombinant protein in yeast

P. pastoris since the protein target will be fused

with a yeast-derived signal sequence factor- . Table

1 shows the hG-CSF protein sequence (isoform-b)

with the peptide linker. The sequence was used as

input and submitted into the DNA Works 3.1 pro-

gram (online) to generate the DNA sequence

needed. The synthetic gene was designed to contain

optimized codon preferences for expression in yeast

P. pastoris. The ORF of the synthetic gene generat-

ed by the program is shown in Table 2.


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Fuad, et.al. 4

Two restriction sites, XhoIand SalI, were added at

both ends of the sequence for cloning purpose in the

yeast expression vector. The stop codon was ex-

cluded since the sequence would be fused with a

poly-His Tag at the C-terminal. However, the stop

codon will be included for the construction of other

version of gene without Tag. The resulting ORF

sequence was 558 bp in length.

Table 1. Polypeptide sequence of CSF3(or hG-CSF) used as input for the synthetic gene

design using the DNA Works 3.1 program.

Polipeptide sequence1 KREAEATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSL

61 GIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFA

121 TTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP

181

Note: The KREAEA sequence is a peptide linker between signal sequence Factor- andthe synthetic gene

Table 2. DNA sequence of the CSF3synthetic gene as output from DNAWorks 3.1.

DNA sequence

1 AAGAGAGAGGCTGAAGCTACTCCACTAGGCCCAGCTTCTTCTTTGCCACAATCTTTTCTT61 TTGAAGTGTTTGGAACAAGTTAGAAAGATTCAGGGTGATGGTGCTGCCTTGCAGGAAAAG

121 TTGTGTGCTACTTACAAGCTGTGTCATCCAGAAGAATTGGTCTTGCTGGGACATTCTTTG

181 GGTATTCCATGGGCTCCATTGTCTTCTTGTCCATCTCAAGCTCTGCAATTGGCTGGTTGT

241 TTGTCTCAGTTGCATTCTGGTTTGTTTCTGTACCAAGGATTGTTGCAAGCTTTGGAAGGT

301 ATTTCTCCAGAGTTGGGACCAACTTTGGATACTTTGCAACTTGATGTTGCTGATTTTGCT

361 ACTACTATTTGGCAACAAATGGAAGAACTAGGTATGGCTCCTGCTTTGCAGCCAACTCAA

421 GGTGCTATGCCAGCCTTTGCATCAGCTTTTCAGAGAAGAGCTGGTGGTGTTTTGGTTGCT

481 TCTCATTTGCAGTCTTTCCTAGAAGTTTCTTACAGAGTTTTGAGACATTTGGCTCAACCA

541

CSF3 Open Reading Frame construction withTBIO method: The DNA Works 3.1 program also

generated 14 oligonucleotides or primers for thegene construction in addition to generating the syn-

thetic gene sequence. Those primers have an aver-

age length of 60 nt (nucleotides). As seen in Fig. 1,

half of them have the sense-strand sequences and

the other half have the antisense-strand sequences

(Primer sequences not shown). Between the adja-

cent primers, there are overlap regions between 18

and 25 nt in length. However, the overlap regions

have been optimized to have equal Tm value (an-

nealing temperature), which is 601oC.

The TBIO-designed primer set was used for thegene synthesis by a PCR-based method. The gene

was synthesized by seven-step sequential inside-

out bidirectional elongation reactions from themiddle to both ends, which are the N- and C-termini

of the synthetic gene sequence. A pair of TBIO pri-

mers was used in each elongation. This method effi-

ciently produced the desired DNA product, which

was the ORF of the synthetic gene as shown in Fig.

2.


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Fig. 1. Construction method of CSF3synthetic gene using the TBIO (thermodynamical-ly balanced inside-out) method. Primer extension was started from the middle part ofthe gene, followed by exterior primer pairs. The process was carried out by sequentialPCR with each pair of primers. Half of the primers have the sensestrand sequence,whereas the other half have the anti-sensestrand sequence.

Fig. 2. DNA Analysis of PCR products for the CSF3syn-thetic gene construction using the TBIO method. DNAladder 100pb (lane-1) and PCR products (lane-2 to 8):P7~P8 (lane-2); P6~P9 (lane-3); P5~P10 (lane-4);P4~P11 (lane-5); P3~P12 (lane-6); P2~P13 (lane-7);P1~P14 (lane-8). The synthetic gene final product was558 bp in length (lane-8).

The TBIO-designed primer characteristics were

shown in Table 3 (A-D), including frequency range,

Tm range, overlap length range and primer length

range. The program used to generate the synthetic

gene sequence, the DNA Works 3.1, has a range of

parameters that could be fixed or customized. For

example, when the primer length was set at a fix

value for 60 nt and the Tm range varies from 60 to

65oC, then the program sorted out a number 6 dif-

ferent sequences appropriate for each Tm value.Then one sequence that showed the best Tm range

of 1.3oC was chosen, which was the sequence with

the Tm setting at 60oC. This means that the gap or

difference of annealing temperatures of each adja-

cent primers were not greater than that value

(601.3oC). The less the Tm ranges the better elon-

gation process in the PCR reactions.

The PCR product showed that the sequential

PCR reactions carried out according to this method

worked well (Fig. 2). However, when all of primers

were used in a single PCR mixture and was run in a

single-step PCR, no product was resulted (data notshown). The primer length of 60 nt was chosen for

effective result and cost considerations. Xiong et al.

(2004) has successfully constructed a quiet long

gene sequence called vip3aI (2,382 bp). He reported

the effective cost that should be spent with 60 nt

primer was better than 90 nt primer for the vip3aI

gene synthesis. The cost was reduced to one third

using 60 nt primers compared to 90 nt primers.

p1p2

p3p4

p5p6

p7

p8p9

p10p11

p12p13

p14

Forward primer

Reverse primer

p1p2

p3p4

p5p6

p7

p8p9

p10p11

p12p13

p14

Forward primer

Reverse primer

100

200

300

400

500

1000

600

(bp) 1 2 3 4 5 6 7 8


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Fuad, et.al. 6

Table 3. Some characteristics of the oligonucleotides used for the synthesis of the CSF3synthetic gene. (A) Codon fre-quency range; (B) Annealing temperature range (Tm); (C) Overlap region range; (D) Oligos length.

A B C D

Frequencyrange (%)

No. of

codons

Tm range(oC)

No. of

overlaps

Overlaplength range

(nt)

No. of oligos Length range No. of

oligos

0-4

5-9

10-14

15-19

20-24

25-29

30-34

35-39

40-44

45-49

50

0

0

4

7

2

15

27

14

30

25

56

< 58

58

59

60

61

62

63

64

65

66

0

0

5

8

0

0

0

0

0

0

< 17

17

18

19

20

21

22

23

24

25

26

27

0

0

3

2

3

0

3

1

0

1

0

0

< 49

49-50

51-52

53-54

55-56

57-58

59-60

61-62

63-64

65-66

67-68

69

2

0

0

0

0

0

12

0

0

0

0

0

Cloning and analysis of the synthetic gene: The

PCR-based gene synthesis with TBIO method re-

sulted in a final product of the target gene having

558 bp in length. Each step of the sequential PCR

reactions produced a set of DNA products with in-

cremental length, ranging from around 100 to 558

bp of the final product (Fig. 2). During all amplifi-

cation processes, Pfu DNA polymerase was used to

ensure the accuracy of the amplified DNA sequence

from its primers. However, the Pfu DNA polyme-

rase would produce a blunt end product only. Inview of cloning the synthetic gene product into an

A/T cloning vector, to the DNA product A ove r-

hangs were added at both ends by incubating the

synthetic gene product with Taq DNA polymerase

(at 72oC, for 1 h) prior to ligation process into the

cloning plasmid (pTZ57R/T).

Transformation of the ligation product has success-

fully produced E. coli (strain XL-1 Blue) transfor-

mants which harbor the recombinant plasmid con-

taining the putative synthetic gene sequence. Re-

striction analysis (with XhoIand SalI) was done to

some positive clones obtained and some of them

have shown the correct DNA insert (Figs. 3 and 4).

Fig. 3. Recombinant plasmid miniprep which containsthe CSF3synthetic gene. Lane-1: plasmid w/o DNA in-sert (Control); lane 2-12: different clones of recombinantplasmids.

Fig. 4. Restriction analysis of recombinant plasmidTZ57R-CSF3syn with XhoIand SalI restriction enzymes.

1 2 3 4 5 6 7 8 9 10 11 12 131 2 3 4 5 6 7 8 9 10 11 12 13

1 2 3 4 5 6 7

250

500

7501000

1500

2000

CSF3syn

3000

1 2 3 4 5 6 7

250

500

7501000

1500

2000

CSF3syn

3000

CSF3


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In conclusion, the DNA Works 3.1 program has

enabled everyone to design any DNA sequence or

gene of interest in a flexible manner by combining

methods (TBIO or TBC) and sequence optimization

for a synthetic gene construction. Availability of a

codon usage database referred by this program

might enhance the quality of the DNA sequence

output resulted from this program.

However, regardless of whether codon-optimized

sequences improve the yield of the protein of inter-

est, the TBIO method of PCR-based gene synthesis

provides a forceful alternative approach for engi-

neering DNA sequences for many other uses such as

construction of : (1) predicted genes/ cDNA that are

difficult to clone or the corresponding mRNA

sources are difficult to obtain; (2) alternatively

spliced gene variants; (3) newly designed prokaryo-

tic plasmids that can be used to create new strain ofmicrobes and (4) newly designed eukaryotic vectors

that can be used for transgenic studies, gene therapy

and DNA vaccines.

DNA sequencing of the CSF3 synthetic gene

(CSF3syn) for the sequence analysis is still in

progress. However, restriction analysis has shown

that the target gene having a correct length of

around 558 bp has been successfully synthesized.

The chance of obtaining a fully correct gene se-

quence seems to be high, since the high fidelity Pfu

DNA polymerase is used during the gene construc-

tion. Although there is also possibility to have somemutated sequences, usually point mutation such as

deletion or substitution, it is less expected.

ACKNOWLEDGEMENT

This research is supported by The Indonesian In-

stitute of Sciences (LIPI)s Competitive ResearchProgram 2008, sub-program the Post Genomic Mo-

lecular Farming.

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Http://www.ncbi.nlm.nih.gov/. Colony Stimulating Factor 3

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