CN101698845B - Promoter arising from corynebacterium glutamicum and application thereof - Google Patents
Promoter arising from corynebacterium glutamicum and application thereof Download PDFInfo
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- CN101698845B CN101698845B CN2009102109629A CN200910210962A CN101698845B CN 101698845 B CN101698845 B CN 101698845B CN 2009102109629 A CN2009102109629 A CN 2009102109629A CN 200910210962 A CN200910210962 A CN 200910210962A CN 101698845 B CN101698845 B CN 101698845B
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Abstract
The invention discloses a promoter with a nucleotide sequence as follows: 1) a nucleotide sequence shown in sequence 1 in a sequence table; 2) a nucleotide sequence which hybridizes with the nucleotide sequence in 1) under strict conditions and has promoter activity; or 3) a nucleotide sequence which has homology of more than 70% with the nucleotide sequence in 1) and has promoter activity. The promoter provided by the invention can be applied to the biotechnology field with corynebacterium or escherichia bacteria as industrial microbes. Experiments prove a fragment with promoter activity from SEQ ID NO:1 has the activity higher than that of a trc promoter (SEQ ID NO:2).
Description
Technical field
The present invention relates to biological technical field, particularly have the nucleotide fragments and the application thereof of promoter activity.
Background technology
Corynebacterium is in gram-positive microorganism.Some excellent bacillus are as industrial microorganism widespread use in bioengineering field, can be used for production number of chemical material, for example amino acid and Nucleotide, L-L-glutamic acid, L-Methionin, L-Threonine, L-tryptophane, t-inosinic acid etc.Amino acid and Nucleotide can be used as medicine, food, foodstuff additive, animal-feed.The rod bacillus mutant strain that process physics and/or chemical process mutagenic treatment obtain has the ability of stronger synthetic above-mentioned useful matter; And through genetic engineering and/or metabolic engineering excellent bacillus wild strain or mutant strain are transformed, can obtain to have the more bacterial strain of high production intensity.The elementary operation of this genetic engineering promptly is in excellent bacillus, to express the gene that relates to multiple pathways metabolism.Usually gene all is under promotor control, to express, and its expression intensity depends on promoter element.In plasmid that excellent bacillus gene group, excellent bacillus have and excellent bacillus phage, isolate 33 nucleotide fragments with promoter activity; The promotor that comprises some knowns in these nucleotide fragments for example; ((P á tek M et al. such as amt, argS, fda, gap gene; Journal ofBiotechnology 104:311-323,2003).Promotor from intestinal bacteria, streptomycete and subtilis can be expressed in corynebacterium genus bacteria; Explain that the mRNA polysaccharase in the excellent bacillus both can discern from gram positive bacterium; Can discern again from (the Mart í n J F et al. of the multiple promotor in the gram negative bacterium; Nature Biotechnology 5 (2): 137-146,1987).Still there is function in the operator-repressor system of intestinal bacteria trp operon in Corynebacterium glutamicum, and can be induced by indolylacetic acid (IAA).Tsuchya etc. utilize colibacillary lac and lambda particles phage P
RP
LE.C. 2.3.1.28 (Tsuchiya M & Morinaga Y, NatureBiotechnology 6 (4): 428-430,1988) has been expressed by the operator-repressor system in excellent bacillus.Use tac promotors such as Liebl and lacI have made up derivable intestinal bacteria-Corynebacterium glutamicum shuttle expression carrier; Great expression the nucleicacidase of streptococcus aureus (Liebl W et al.; Journal of Bacteriology 174 (6): 1854-1861,1992).Known trc promotor can show active (Patek M et al., Journal ofBiotechnology 104:311-323,2003) in corynebacterium genus bacteria.The expression system that in Corynebacterium glutamicum, makes up has mostly used the strong promoter in the intestinal bacteria, for example Ptrp, PlacUV5, Ptac etc.
But, check because colibacillary promotor has to leak in excellent bacillus, and relevant inductor is difficult for passing the reasons such as cell walls of excellent bacillus under low concentration, thereby its application is also very limited in actual production.Still need from Corynebacterium glutamicum, screen and to receive the rigorous promotor of various physical chemical factor inductive.
Summary of the invention
The object of the present invention is to provide a kind of promotor.
Promotor provided by the invention is from the nucleotide fragments that obtains and have promoter activity from the Corynebacterium glutamicum gene component.Term among the present invention " promotor " means DNA the preceding paragraph sequence, and RNA polymerase is attached on this sequence, can initial gene transcribe.
The nucleotide sequence of said promotor is following 1) or 2) or 3):
1) nucleotide sequence shown in the sequence 1 in the sequence table;
2) under the rigorous condition of height with said 1) nucleotide sequence hybridization and have the nucleotide sequence of promoter activity;
3) with said 1) nucleotide sequence have the homology more than 70% and have the nucleotide sequence of promoter activity.
The rigorous condition of said height is meant, with Hybond membrane place prehybridization solution (the 0.25mol/L sodium phosphate buffer, pH7.2,7%SDS) in, 65 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.2,7%SDS, isotope-labeled nucleotide fragments), 65 ℃ of hybridization 12hr; Abandon hybridization solution, (20mmol/L sodium phosphate buffer, pH7.2 5%SDS), wash film 2 times for 65 ℃, each 30min to add film washing liquid I; (20mmol/L sodium phosphate buffer, pH7.2 1%SDS), wash film 30min for 65 ℃ to add film washing liquid II.
Promoter active fragment of the present invention also comprise with from the segmental nucleotide sequence complementary nucleotide sequence of SEQ ID NO.1.Term used herein " complementary " mean follow that basepairing rule produces meaning.
Those of ordinary skills can adopt known method at an easy rate, and for example the method for orthogenesis and point mutation is suddenlyd change to promotor nucleotide sequence of the present invention.Those are through manually modified; Have and separate the promotor nucleotide sequence 70% that obtains or the Nucleotide of higher homology with the present invention; As long as kept expressing the promoter activity of target gene, all be to be derived from nucleotide sequence of the present invention and to be equal to sequence of the present invention.
Term used herein " homology " refers to the sequence similarity with the natural acid sequence." homology " comprises with promotor nucleotide sequence of the present invention having preferably 75% or higher, more preferably 85% or higher, even more preferably 90% or higher, and most preferably 95% or the nucleotide sequence of higher identity.Homology can be with the naked eye or computer software estimate.The software that uses a computer, the homology between two or more sequences can be used per-cent (%) expression, and it can be used for estimating the homology between the correlated series.
The recombinant vectors that contains above-mentioned promotor also belongs within protection scope of the present invention.
Above-mentioned recombinant vectors is that above-mentioned promotor is inserted the recombinant vectors that carrier pAKC6 MCS is processed.
The transgenic cell line that contains above-mentioned promotor also belongs within protection scope of the present invention.
The reorganization bacterium that contains above-mentioned promotor also belongs within protection scope of the present invention.
Above-mentioned reorganization bacterium is the protokaryon bacterium that contains above-mentioned promotor, and said protokaryon bacterium is following 1) or 2) or 3):
1) corynebacterium genus bacteria
2) Escherichia bacterium
3) mutant strain that obtains through mutagenesis of Corynebacterium or Escherichia bacterium.
Promoter active fragment of the present invention also has activity except in corynebacterium genus bacteria, having the activity in the Escherichia bacterium.The corynebacterium genus bacteria of indication can be any bacterium of this genus among the present invention, and for example Corynebacterium glutamicum ATCC 13032, Corynebacterium crenatum AS 1.542 and Beijing rod bacillus AS 1.299 or the like.But corynebacterium genus bacteria is not limited thereto, and also comprises the mutant strain that is obtained through method mutagenesis various physics or chemistry by corynebacterium genus bacteria.The Escherichia bacterium of indication comprises intestinal bacteria among the present invention.
The promotor that another object of the present invention is to provide above-mentioned starts the application in the destination gene expression in Corynebacterium or Escherichia bacterium.
The above-mentioned purpose gene can be chloramphenicol acetyl transferasegene.
The reorganization rod bacillus or the Escherichia that contain above-mentioned promotor can be produced amino acid and Nucleotide etc. through routine techniques, can be used as medicine, food, foodstuff additive or animal-feed like L-L-glutamic acid, L-Methionin, L-Threonine, L-tryptophane, t-inosinic acid etc.
The experiment proof: the promoter active fragment from SEQ ID NO:1 has in corynebacterium genus bacteria than the higher activity of trc promotor (SEQ ID NO:2).
Description of drawings
Fig. 1 is a promoter active fragment in shotgun cloning Corynebacterium glutamicum 10147 genomes.
Fig. 2 is the promoter probe vector design of graphics that has the trc promotor.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but the present invention is not limited to following examples.
Among the following embodiment,, be ordinary method like no specified otherwise.
Term " tac " starts subsystem with-35 districts of the promotor of intestinal bacteria tryptophan operon and-10 districts of intestinal bacteria lec operon, and operation Gene Partial in the lec operon and the fusion of SD sequence form, and-35 districts are interval at a distance from 16 bases with-10.Known tac promotor is very strong promotor.Term " trc " promotor ties up to be inserted a base and makes up and obtain between-35 districts and-10 districts of " tac " promotor, its-35 district and-10 districts are spaced apart 17 bases.Known in intestinal bacteria the activity of trc promotor be 90% of tac promotor.(Brosius?J?et?al.，The?Journal?of?Biological?Chemistry 260(6)：3539-3541)。
The LB substratum: every liter of substratum contains Tryptones 10g, yeast powder 5g, and NaCl 10g is settled to 1 liter with zero(ppm) water.
Embodiment 1, acquisition have the dna fragmentation of promoter activity
One, cultivates Corynebacterium glutamicum 10147 and extract genomic dna
1, the cultivation of bacterium
Picking Corynebacterium glutamicum 10147 (bar-shaped bacterioid electric shock transform in multiple condition transformation efficiency influenced biotechnology journal 11 (3) such as Shen Tian Xiang: 245-279 1995) (this bacterial strain can obtain in Institute of Microorganism, Academia Sinica; Specifically see the letter of guarantee) single colony inoculation is in the test tube of 3ml LB substratum; Place 300rpm incubated overnight on 30 ℃ the shaking table, this nutrient solution is as seed liquor.Get seed liquor 400 μ L and be inoculated in the triangular flask of 30ml LB substratum, place 300rpm incubated overnight on 30 ℃ the shaking table.
2, the extraction of Corynebacterium glutamicum 10147 genomic dnas
The centrifugal 5min of 10000rpm collects the thalline of incubated overnight in the step 1, and (50mmol/L glucose, 25mmol/L Tris-HCl pH8.0,10mmol/L EDTA pH8.0) washes bacterium twice with the 5ml solution I.Thalline suspends with the 4.5ml solution I, and adding N,O-Diacetylmuramidase to its final concentration is 10-15mg/ml, and suspension-s is handled 1-2h in 37 ℃.Add 0.5ml10%SDS, the vibration mixing is to bright.Add 1ml 5M NaCl then, place 1h in 4 ℃.The thermal agitation centrifuge tube repeatedly is creamy white to solution.13500rpm, 4 ℃ of centrifugal 30min.The imitative extracting of equal-volume phenol 3 times, 14000rpm, centrifugal 10min gets supernatant.Add 2 times of volume absolute ethyl alcohols, stir out thread deposition with glass stick, this deposition promptly is a genomic dna.Use 70% washing with alcohol one time then, will precipitate and be dissolved among the 1ml TE (10mM Tris-HClpH8.0,1mM EDTA pH8.0) after air-dry, add suitable RNase and handle RNA.
Two, screening obtains promoter fragment
Adopt shotgun, utilize the promotor carrier detection from Corynebacterium glutamicum 10147 genomic dnas, to screen the dna fragmentation with promoter activity, the process of screening promotor is referring to Fig. 1.
Genomic dna with restriction endonuclease Sau3A I complete degestion Corynebacterium glutamicum 10147; With Sau3A I fragment and promotor carrier detection pAKC6 (the structure Li Kai of Corynebacterium glutamicum/bacillus coli shuttle type promoter probe vector etc. that cut through restriction endonuclease Bgl II enzyme; Mikrobe journal 200747 (2): 191-196) connect (Sau3A I and Bgl II belong to isocaudarner); Transformed into escherichia coli DH5 α competent cell (Sambrook J, Fritsch E, Maniatis T molecular cloning experiment guide. Jin Dongyan; Li Mengfeng; Deng translating. second edition. Beijing: Science Press, 1989), conversion fluid is coated the LB flat board that contains 60 μ g/mL paraxin and is carried out primary dcreening operation; After 37 ℃ of incubators are cultivated 24h, carry out multiple sieve with the dibbling of toothpick picking transformant in the LB flat board that contains 400 μ g/mL paraxin, cultivate 24h in 37 ℃ of incubators; From the transformant bacterium colony that can grow, extract plasmid, use SEQ ID NO:3 and 4 primers to carry out the PCR response analysis, reaction conditions: 94 ℃ of 2min; 94 ℃ of 30s, 55 ℃ of 1min, 72 ℃ of 3min, 30 circulations; 72 ℃ of 10min.Picking inserts the transformant of fragment less than 600bp; From these transformants, extract competent cell (the Liebl W et al. that the plasmid electric shock transforms Corynebacterium glutamicum 10147 then; Brosius J et al., FEMS Microbiology Letters, 65 (3): 299-304; 1989), conversion fluid is coated the LB flat board that contains 50 μ g/mL kantlex with the screening transformant.The transformant dibbling is had the segmental transformant of insertion in the LB flat board that contains 30 μ g/mL paraxin with screening, cultivate 24h, from the bacterium colony that can grow, extract plasmid, carry out the PCR response analysis once more in 30 ℃ of incubators.Be that same operation is carried out in contrast simultaneously with pAKC6.As a result, obtain 30 active fragmentss with promoter function.This result shows, in intestinal bacteria and corynebacterium genus bacteria, all shows active according to the promoter active fragment of embodiment of the present invention.
Embodiment 2, check and analysis promoter fragment
One, the determined dna sequence of promoter active fragment
Extract 30 DNAs among the embodiment 1 (two), use artificial synthetic primer SEQ ID NO:3 to carry out the determined dna sequence of promoter active fragment respectively, obtain 30 dna sequence dnas with active fragments of promoter function.The promoter sequence that this patent relates to is SEQ ID NO:1.
Two, the mensuration of total protein content
1) preparation of crude enzyme liquid
Dna fragmentation shown in the preparation sequence SEQ ID NO:1.
This fragment is connected with the promotor carrier detection pAKC6 that cuts processing through restriction endonuclease Bgl II enzyme, adopts Calcium Chloride Method (Sambrook J, Fritsch E; Maniatis T molecular cloning experiment guide. Jin Dongyan; Li Mengfeng waits and translates. second edition. and Beijing: Science Press, 1989) change bacillus coli DH 5 alpha over to; Screening positive clone uses primer SEQ ID NO:3 and SEQ ID NO:4 to carry out PCR, order-checking evaluation.Sequencing result shows: the purpose fragments sequence of amplification is identical with SEQ ID NO:1.
Extract the DNA of above-mentioned positive colony, electric shock transforms the competent cell of Corynebacterium glutamicum 10147, conversion fluid is coated on the LB flat board that contains 30 μ g/mL paraxin screened transformant then; Extract plasmid, after PCR identifies, with identifying that the single colony inoculation of Corynebacterium glutamicum after correct is in liquid LB substratum; Place 30 ℃ of shaking tables to cultivate 16h; The centrifugal 5min of 10000rpm collects thalline, with 100mmol/L Tris-HCl (pH7.8) washing twice, suspends.Ultrasonic disruption cell (condition: liquor capacity 300 μ L, power 280w, work 10s, interval 30s, 30 circulations), in 4 ℃, the centrifugal 15min of 13500rpm gets the mensuration that supernatant is used for crude enzyme liquid.
With the Corynebacterium glutamicum that has pAKC6 and pAKC6-trc is contrast, carries out same experiment.
Wherein pAKC6-trc is promotor trc and being connected of pAKC6, and connection procedure is referring to Fig. 2, and concrete steps are following:
With reference to plasmid pXC99E (Kirchner O & Tauch A, Journal Biotechnology, 104 (1-3): 287-299; 2003) dna sequence dna; Synthetic promotor trc adds the restriction enzyme site of KpnI and Hind III respectively in its both sides when synthetic, sequence is seen SEQ ID No.2.This fragment is connected with promotor carrier detection pAKC6 through restriction endonuclease Kpn I and HindIII double digestion, and electric shock transforms the competent cell of Corynebacterium glutamicum 10147, and the recombinant vectors pAKC6-trc that obtains containing known promotor trc is as positive control.
2) mensuration of total protein content
Adopting Xylene Brilliant Cyanine G method (Bradford M, Analytical Biochemistry, 72 (1-2): 248-254,1976), is standard protein with bovine serum albumin (component V).
The albumen colouring reagents: take by weighing 100mg Xylene Brilliant Cyanine G G-250 and be dissolved in 50ml 95% ethanol, add 100ml 85% phosphoric acid then, last constant volume is to 1L, and is subsequent use with in brown bottle, preserving behind the filter paper filtering.
Protein standard solution: take by weighing 25mg bovine serum albumin (component V) and be dissolved in the standardized solution that is mixed with 1mg/ml in the 25ml zero(ppm) water.
Typical curve: measure 100 μ L protein solutions (protein standard solution and the zero(ppm) water of 1mg/ml are formed in varing proportions) and 5ml albumen colouring reagents mixing, measure OD after leaving standstill 5min
595, according to measured value production standard curve.
Measure: get earlier 10 μ L testing samples with 90 μ L distilled water dilutings to 0.1ml, be added in the test tube, add 5ml albumen colouring reagents then, vibration shakes up, and measures OD after leaving standstill 5min
595
Mensuration result is as shown in table 1, and it is lower slightly to contain the total protein concentration that Corynebacterium glutamicum that total protein concentration ratio that the Corynebacterium glutamicum of sequence 1 gives expression to contains sequence 2 (trc promotor) gives expression to.
Insert segmental size and promoter activity in the table 1. Corynebacterium glutamicum transformant
Two, the active mensuration of E.C. 2.3.1.28 (CAT)
Through the mensuration that the reporter gene E.C. 2.3.1.28 enzyme on the promotor carrier detection pAKC6 is lived, promoter active fragment more of the present invention and be usually used in the activity of trc promotor in Corynebacterium glutamicum 10147 in the Corynebacterium glutamicum.
Reference method (Shaw W V, Method in Enzymology, 43:737-755; 1975) carry out, the 1.0ml reaction system contains 100mM Tris-HCl (PH7.8), 0.1mM acetyl-CoA; 0.4mg/ml 5,5 '-two sulphur (2-nitrobenzoic acid) [DTNB], the crude enzyme liquid of an amount of volume; Reaction mixture is heated to 37 ℃ in water-bath after, adding paraxin, to make its final concentration be 0.1mM, and mixing is measured OD immediately
412With the reaction solution that does not add paraxin is contrast.Each sample determination three times is averaged and is made enzyme curve alive.
The definition of CAT activity unit: a unit of activity (U) is under above-mentioned reaction conditions, the required enzyme amount of PM acetylize 1 μ mol paraxin.
The result is as shown in table 1, according to embodiments of the present invention promoter active fragment can be in coryneform bacterium the effective expression chloramphenicol acetyl transferasegene.Particularly, SEQ ID NO:1 promoter active fragment demonstrates high reactivity in excellent bacillus, and the activity of this specific activity trc promotor (SEQ ID NO:2) is higher.
Sequence table
< 110>Institute of Microorganism, Academia Sinica
< 120>promotor of derived from corynebacterium glutamicum and application thereof
<130>CGGNAL92649
<160>4
<210>1
<211>437
<212>DNA
< 213>Corynebacterium glutamicum (Corynebacterium glutamicum)
<400>1
gatccgctgg?aaacccgaca?agccgtattg?gccgtcaaag?actggattga?aggggaggga 60
gacgtcgaaa?agcctggtcg?tgcggcgctt?gccgccgcaa?ctcgcctgag?cgtccgactg 120
ctcgcgcaag?acgcgccggg?aaacagcgtg?gaggtgcggg?tacccccatt?tgttgcggtg 180
caatgcatag?aggggccaaa?acatacacgc?ggcacaccac?ccaacgtggt?ggagaccgac 240
gccaagacct?ggttacgctt?agcaactggg?caaaccacat?ttgatgcaga?atttgaaagc 300
ggaaaaatta?gcgcatcagg?tacccgagcc?aaagagattg?cggactggtt?accagtggtc 360
aaactttaga?tttcctaatg?ctcattagtt?tctgggcaac?cgacagacta?cgatttgacc 420
tgtggcatta?cttgatc 437
<210>2
<211>295
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
ggtaccgcga?attgatctgg?tttgacagct?tatcatcgac?tgcacggtgc?accaatgctt 60
ctggcgtcag?gcagccatcg?gaagctgtgg?tatggctgtg?caggtcgtaa?atcactgcat 120
aattcgtgtc?gctcaaggcg?cactcccgtt?ctggataatg?ttttttgcgc?cgacatcata 180
acggttctgg?caaatattct?gaaatgagct?gttgacaatt?aatcatccgg?ctcgtataat 240
gtgtggaatt?gtgagcggat?aacaatttca?cacaggaaac?agaccatgga?agctt 295
<210>3
<211>25
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>3
aaaaggtgtc?aacctcgata?atttg 25
<210>4
<211>25
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>4
gaacggtctg?gttataggta?cattg 25
Claims (7)
1. promotor, its nucleotide sequence is shown in sequence in the sequence table 1.
2. the recombinant vectors that contains the described promotor of claim 1.
3. recombinant vectors according to claim 2 is characterized in that: said recombinant vectors is the recombinant vectors that the Bgl II site of the described promotor insertion of claim 1 carrier pAKC6 is processed.
4. the transgenic cell line that contains the described promotor of claim 1.
5. the reorganization bacterium that contains the described promotor of claim 1.
6. reorganization bacterium according to claim 5 is characterized in that: said reorganization bacterium is the protokaryon bacterium that contains the said promotor of claim 1, and said protokaryon bacterium is following 1) or 2):
1) corynebacterium genus bacteria,
2) Escherichia bacterium.
7. the described promotor of claim 1 starts the application in the chloramphenicol acetyl transferasegene expression in Corynebacterium glutamicum 10147 or bacillus coli DH 5 alpha.
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| CN106434652B (en) * | 2016-07-29 | 2020-05-22 | 江南大学 | Exogenous promoter suitable for corynebacterium glutamicum and application thereof |
| EP3604530A4 (en) | 2017-03-21 | 2020-11-25 | Wuhan Grand Hoyo Co., Ltd. | Corynebactetium constitutive expression vector promoter screened on the basis of transcriptome sequencing, screening method therefor, and applications thereof |
| CN107142234B (en) * | 2017-05-12 | 2020-09-15 | 清华大学 | Method for producing tetrahydropyrimidine by utilizing fermentation of recombinant corynebacterium glutamicum |
| CN109097361B (en) * | 2018-08-28 | 2020-02-14 | 江南大学 | Promoter, vector thereof and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250548A (en) * | 2007-03-30 | 2008-08-27 | 中国科学院微生物研究所 | Shuttle plasmid and derivative plasmid thereof |
| WO2009025470A3 (en) * | 2007-08-17 | 2009-04-16 | Cj Cheiljedang Corp | Novel corynebacterium glutamicum promoter |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250548A (en) * | 2007-03-30 | 2008-08-27 | 中国科学院微生物研究所 | Shuttle plasmid and derivative plasmid thereof |
| WO2009025470A3 (en) * | 2007-08-17 | 2009-04-16 | Cj Cheiljedang Corp | Novel corynebacterium glutamicum promoter |
Non-Patent Citations (2)
| Title |
|---|
| Miroslav pàtek.Function of Corynebacterium promoters in Escherichia coli,Streptomyces lividans, and Bacillus subtilis..《Journal of Biotechnology》.2003,第104卷(第1-3期),325-334. * |
| 刘桂明等.谷氨酸棒杆菌10147基因组中启动子活性片段的克隆与分析..《微生物学报》.2009,第49卷(第7期),摘要,材料与方法,图1,表2. * |
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| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120502 Termination date: 20191112 |