CN106591310B - A kind of Escherichia coli high-strength combination promoter and its application - Google Patents
A kind of Escherichia coli high-strength combination promoter and its application Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
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Abstract
The invention discloses a kind of Escherichia coli high-strength combination promoter and its applications, belong to synthesising biological technical field.The present invention provides high Intensity of Transcription of Endothelial promoter PssrA、PdnaKJ、PgrpE、PalsRBACEIntensity promoter P is translated with heightrpsT.Respectively by PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsT4 kinds of combination promoters with high expression intensity are obtained after fusion.The maximum intensity of the combination promoter is the P of 10mM arabinose inductionBAD1.7 times of promoter.The combination promoter is a kind of safety and efficient gene expression element.
Description
Technical field
The present invention relates to a kind of Escherichia coli high-strength combination promoter and its applications, belong to synthesising biological technical field.
Background technique
As first control element of gene expression, the table of gene downstream is controlled by regulating and controlling the intensity of promoter
Become most direct effective means up to level.During metabolic engineering, the strategy such as modularization utilizes different copy numbers
Plasmid and IPTG induction promoter successfully multiple gene expression doses of metabolic pathway are finely tuned, greatly increase
Purpose product yield is added.
Currently, the most commonly used is a series of inducible promoters in Escherichia coli, however due to inducible promoter
Quantity limitation, therefore these promoters are often difficult to and carry out accuracy controlling to each Gene expression intensities.Microorganism
A large amount of natural promoter is carried on genome, these promoter intensity multiplicity have applied to the huge of metabolic engineering
Big potentiality.However, since the intensity of most of constitutive promoter is unknown, and promoter precise sequence corresponding with intensity is not
Know so that directly difficult using constitutive promoter progress gene expression.Furthermore the transcription of most natural promoter is strong
The translation intensity disunity in gene is spent, the protein that often promoter with high transcriptional level is translated is not but high, and low
The promoter of transcriptional level but possesses higher protein expression level.It often multiple promoters, enhancer and declines on genome
Subtract the expression that the elements such as son adjust a gene jointly, therefore the promoter region of different length its function is also not quite similar.Such as
What, which finds the constitutive promoter with high Intensity of Transcription of Endothelial and high translation intensity, becomes key.
Summary of the invention
To solve the above-mentioned problems, it present invention firstly provides one group of Escherichia coli constitutive promoter, clearly determines
Its Intensity of Transcription of Endothelial and translation intensity, provide its specific DNA sequence dna.The present invention is the inducer during avoiding gene expression
Addition provides selection, while can be used for adjusting polygenic expression by way of control bacterial metabolism flow direction.
Shown in the nucleotide sequence of the Escherichia coli constitutive promoter such as NO.6~9 SEQ ID are any.
The present invention also provides a kind of expression vector, have nucleotide sequence as NO.6~9 SEQ ID it is any shown in start
Son.The skeleton of the expression vector includes the expression vector of common Escherichia coli.
The present invention provides the recombinant bacterium of application promoter building.
In one embodiment of the invention, the host of the recombinant bacterium includes various existing Bacillus coli expression places
It is main.
In one embodiment of the invention, the recombinant bacterium constructed using the promoter is to utilize the starting
Son connection target gene, construction recombination plasmid, and express in host.
In traditional genetic engineering research, inducible promoter is frequently used to expression related gene, however due to needing
It adds inducer and is difficult to realize simultaneously be finely adjusted multiple Gene expression intensities, inducible promoter is dfficult to apply to more
Gene Expression Pathway.Constitutive promoter from genome is often due to its expression intensity is lower is equally difficult to meet to want
It asks.The 4 kinds provided by the invention high-strength combination promoters with gradient intensity do not need additional addition inducer, have height
Intensity of Transcription of Endothelial and high translation intensity, while it can be used for pair with certain intensity gradient (promoter intensity span is 7.7 times)
Multi-gene expression approach is finely adjusted.
Specific embodiment
Materials and methods
E. coli jm109 is used for plasmid construction, and e. coli k12 MG1655 is strong for expressing protein determination promoter
Degree.
Embodiment 1
Escherichia coli transcript profile data are analyzed, using e. coli k12 MG1655 genome as template, expand promoter
PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsT.For the intensity for measuring this 5 promoters, P is merged in a manner of fusion DNA vaccinessrA、
PdnaKJ、PgrpE、PalsRBACE、PrpsTWith reporter gene EGFP.
To measure and constructing high-strength combination promoter, by promoter P in a manner of fusion DNA vaccinessrA、PdnaKJ、PgrpEWith
PalsRBACETranscriptional control regions (- 10th area and -35th area) and promoter PrpsTThe region 5'-UTR fusion, thus building obtain 4
Kind combination promoter PssrA-rpsT、PdnaKJ-rpsT、PgrpE-rpsTAnd PalsRBACE-rpsT.Then will combination promoter and EGFP gene with
The mode of fusion DNA vaccine is merged for measuring combination promoter intensity.Pcr amplification reaction is by high-fidelity DNA polymerase
PrimeSTAR HS (premix) (TaKaRa) amplification.Amplimer is shown in Table 1.
1 the primer sequence of table
Artificial sequence
Fusion DNA vaccine product is connect after BamHI/SacI double digestion with the pCDFDuet-1 plasmid of same double digestion,
In chemical conversion e. coli k12 MG1655.Correct bacterial strain is sequenced for subsequent promoter strength detection.
1, Intensity of Transcription of Endothelial
Recombinant bacterium is cultivated under the conditions of 220rpm 37 DEG C in LB culture medium or MOPS, culture to after stationary phase (11h) in
4000rpm, 4 DEG C thalline were collected by centrifugation, extracts total mRNA of thallus, and real-time quantitative PCR detection is carried out after reverse transcription, measures EGFP
MRNA abundance intracellular, testing result is shown, using promoter PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsT, mRNA abundance
It is the P of 10mM arabinose induction respectivelyBAD46.3,25.6,8.3,6.4 and 0.0047 times of promoter.
2, intensity is translated
In order to measure constitutive promoter PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsTTranslation intensity, using EGFP as table
Up to gene, stationary phase detection detection EGFP intracellular intracellular is horizontal in Escherichia coli MG1655.Recombinant bacterium is cultivated in LB or MOPS
It 37 DEG C in base, is cultivated under the conditions of 220rpm, in 4000rpm after culture to stationary phase (11h), 4 DEG C thalline were collected by centrifugation, utilizes
PBS (pH=7.4) buffer solution for cleaning cell 2 times, to remove dead thallus, thallus secretion and culture medium precipitate component to reduce
Fluorescence detection background.By the thallus after cleaning as in 96 hole fluorescent plates in Multifunction fluorescent microplate reader fluorescence intensity and bacterium
Body OD600, testing conditions are 488nm excitation, 520nm transmitting.It is fluorescence intensity and OD that promoter, which translates intensity,600Ratio.
Fluorescence microplate reader testing result shows, promoter PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsTTranslation intensity point
Not Wei 10mM arabinose induction PBAD0,0.16,0.06,0.016 and 0.26 times of promoter.
Compare promoter PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsTTranscription and translation intensity discovery to EGFP, starting
Sub- PssrA、PdnaKJ、PgrpEAnd PalsRBACEIntensity of Transcription of Endothelial with higher, and it is relatively low to translate intensity.Promoter PrpsTHave
Lower Intensity of Transcription of Endothelial, and it is higher to translate intensity.Analyze reason, it may be possible to due to promoter PssrA、PdnaKJ、PgrpEAnd PalsRBACE
The promoter P containing strong transcriptional control regions (- 10th area and -35th area)rpsTThen there is the stronger region 5'-UTR.Therefore it combines
Both sides' advantage is possible to building and obtains high-intensitive combination promoter
3, the translation intensity of promoter is combined
In order to measure the combination promoter P after promoter is engineeredssrA-rpsT、PdnaKJ-rpsT、PgrpE-rpsTWith
PalsRBACE-rpsTExpression intensity, using EGFP as expressing gene, stationary phase detects detection born of the same parents intracellular in Escherichia coli MG1655
Interior EGFP is horizontal.The translation intensity of fluorescence microplate reader testing result display combination promoter is respectively what 10mM arabinose induced
PBAD1.70,1.37,0.80 and 0.22 times of promoter.It follows that the four kinds of combination promoters obtained by promoter engineering
PssrA-rpsT、PdnaKJ-rpsT、PgrpE-rpsTAnd PalsRBACE-rpsTIt has been provided simultaneously with PssrA、PdnaKJ、PgrpEAnd PalsRBACEThe height of promoter
Transcriptional efficiency and PrpsTThe high translation efficiency of promoter.
4, the universality of promoter expressing gene is combined
Since four kinds of combination promoters all have the identical region 5'-UTR, in order to detect this combination tandem promoter
Sub- 5'-mRNA can only choose promoter P for the ability to express of different genesssrA-rpsTOther reporter genes are expressed, and
Its expression intensity is measured, so that it is determined that deriving from PrpsT5'-mRNA influence that different genes are expressed.
P is used respectivelyssrA-rpsTIt combines promoter and merges table with red fluorescent protein (RFP) and beta galactosidase (LacZ)
It reaches, then measuring it and translating intensity is respectively PBAD1.4 times of promoter and 1.7 times.It is demonstrated experimentally that being directed to different purpose bases
Cause combines promoter expression intensity equally with higher.
5, the negative sense result of combination promoter building
It should be noted that former due to the influence of mRNA secondary structure, and to promoter can be destroyed when starting sub-portfolio transformation
Some functional areas, therefore the transcript regions of the high-intensitive promoter of combination and the region 5'-UTR may not be able to access forward junction
Fruit.
Utilize another high translation intensity constitutive promoter PinfC-rplTThe region 5'-UTR and PssrA、PdnaKJ、PgrpE、
PalsRBACETranscript regions combination construct a series of combination promoter, PssrA-infC-rplT、PdnaKJ-infC-rplT、PgrpE-infC-rplT
And PalsRBACE-infC-rplT.However, being measured discovery to the translation intensity of these four combination promoters: it translates intensity difference
For PinfC-rplT0.94,0.58,0.51,0.21 times.These types combination promoter intensity is declined.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of Escherichia coli high-strength combination promoter and its application
<160> 23
<170> PatentIn version 3.3
<210> 1
<211> 146
<212> DNA
<213>e. coli k12 MG1655
<400> 1
attggctatc acatccgaca caaatgttgc catcccattg cttaatcgaa taaaaatcag 60
gctacatggg tgctaaatct ttaacgataa cgccattgag gctggtcatg gcgctcataa 120
atctggtata cttaccttta cacatt 146
<210> 2
<211> 175
<212> DNA
<213>e. coli k12 MG1655
<400> 2
gcacaaaaaa tttttgcatc tcccccttga tgacgtggtt tacgacccca tttagtagtc 60
aaccgcagtg agtgagtctg caaaaaaatg aaattgggca gttgaaacca gacgtttcgc 120
ccctattaca gactcacaac cacatgatga ccgaatatat agtggagacg tttag 175
<210> 3
<211> 99
<212> DNA
<213>e. coli k12 MG1655
<400> 3
gattgatgac aatgtgagtg cttcccttga aaccctgaaa ctgatcccca taataagcga 60
agttagcgag atgaatgcga aaaaaacgcg gagaaattc 99
<210> 4
<211> 206
<212> DNA
<213>e. coli k12 MG1655
<400> 4
agcaacatct atcatctaaa aaaccagaaa aacaaataac atcatgtttt taaactaatt 60
aaatgaaata aaattttaag ccactcgcca ttgttcacaa taaaataaac tttataaatt 120
ttattttttt gtgaagtcgc cagcatcttt tctgttcttg ctgtggtgat atagtggcgt 180
cttcaattca aggacaagag aacgtg 206
<210> 5
<211> 192
<212> DNA
<213>e. coli k12 MG1655
<400> 5
tgctgcaatt tttatcgcgg aaaagctgta ttcacacccc gcaagctggt agaatcctgc 60
gccatcacta cgtaacgagt gccggcacat taacggcgct tatttgcaca aatccattga 120
caaaagaagg ctaaaagggc atattcctcg gcctttgaat tgtccatata gaacacattt 180
gggagttgga cc 192
<210> 6
<211> 271
<212> DNA
<213>artificial sequence
<400> 6
attggctatc acatccgaca caaatgttgc catcccattg cttaatcgaa taaaaatcag 60
gctacatggg tgctaaatct ttaacgataa cgccattgag gctggtcatg gcgctcataa 120
atctggtata cttaccttta ccatcactac gtaacgagtg ccggcacatt aacggcgctt 180
atttgcacaa atccattgac aaaagaaggc taaaagggca tattcctcgg cctttgaatt 240
gtccatatag aacacatttg ggagttggac c 271
<210> 7
<211> 288
<212> DNA
<213>artificial sequence
<400> 7
gcacaaaaaa tttttgcatc tcccccttga tgacgtggtt tacgacccca tttagtagtc 60
aaccgcagtg agtgagtctg caaaaaaatg aaattgggca gttgaaacca gacgtttcgc 120
ccctattaca gactcacaac cacatgatga ccgaatacca tcactacgta acgagtgccg 180
gcacattaac ggcgcttatt tgcacaaatc cattgacaaa agaaggctaa aagggcatat 240
tcctcggcct ttgaattgtc catatagaac acatttggga gttggacc 288
<210> 8
<211> 192
<212> DNA
<213>artificial sequence
<400> 8
gattgatgac aatgtgagtg cttcccttga aaccctgaaa ctgatcccca taataagcga 60
accatcacta cgtaacgagt gccggcacat taacggcgct tatttgcaca aatccattga 120
caaaagaagg ctaaaagggc atattcctcg gcctttgaat tgtccatata gaacacattt 180
gggagttgga cc 192
<210> 9
<211> 197
<212> DNA
<213>artificial sequence
<400> 9
agcaacatct atcatctaaa aaaccagaaa aacaaataac atcatgtttt taaactaatt 60
aaatgaccat cactacgtaa cgagtgccgg cacattaacg gcgcttattt gcacaaatcc 120
attgacaaaa gaaggctaaa agggcatatt cctcggcctt tgaattgtcc atatagaaca 180
catttgggag ttggacc 197
<210> 10
<211> 40
<212> DNA
<213>artificial sequence
<400> 10
ctagctagct agggatccat tggctatcac atccgacaca 40
<210> 11
<211> 56
<212> DNA
<213>artificial sequence
<400> 11
tgaaaagttc ttctccctta cccataatgt gtaaaggtaa gtataccaga tttatg 56
<210> 12
<211> 51
<212> DNA
<213>artificial sequence
<400> 12
ggcactcgtt acgtagtgat ggtaaaggta agtataccag atttatgagc g 51
<210> 13
<211> 41
<212> DNA
<213>artificial sequence
<400> 13
ctagctagct agggatccgc acaaaaaatt tttgcatctc c 41
<210> 14
<211> 52
<212> DNA
<213>artificial sequence
<400> 14
tgaaaagttc ttctccctta cccatctaaa cgtctccact atatattcgg tc 52
<210> 15
<211> 45
<212> DNA
<213>artificial sequence
<400> 15
ggcactcgtt acgtagtgat ggtattcggt catcatgtgg ttgtg 45
<210> 16
<211> 34
<212> DNA
<213>artificial sequence
<400> 16
tagggatccg attgatgaca atgtgagtgc ttcc 34
<210> 17
<211> 47
<212> DNA
<213>artificial sequence
<400> 17
tgaaaagttc ttctccctta cccatgaatt tctccgcgtt tttttcg 47
<210> 18
<211> 37
<212> DNA
<213>artificial sequence
<400> 18
atagggatcc agcaacatct atcatctaaa aaaccag 37
<210> 19
<211> 49
<212> DNA
<213>artificial sequence
<400> 19
tgaaaagttc ttctccctta cccatcacgt tctcttgtcc ttgaattga 49
<210> 20
<211> 29
<212> DNA
<213>artificial sequence
<400> 20
tagggatccg cgggcattcg tgttaaagc 29
<210> 21
<211> 52
<212> DNA
<213>artificial sequence
<400> 21
tgaaaagttc ttctccctta cccatacctt attcctccaa ttgtttaaga ct 52
<210> 22
<211> 34
<212> DNA
<213>artificial sequence
<400> 22
cgcggtaccg agctcggccg caaattaaag cctt 34
<210> 23
<211> 24
<212> DNA
<213>artificial sequence
<400> 23
atgggtaagg gagaagaact tttc 24
Claims (7)
1. a kind of element for controlling gene expression, which is characterized in that shown in its nucleotide sequence such as NO.6~9 SEQ ID are any.
2. the element of the control gene expression of one group of gradient intensity, which is characterized in that including nucleotide sequence such as SEQ ID NO.6
At least two in promoter shown in~9.
3. a kind of expression vector, which is characterized in that the nucleotide sequence of its gene expression control elements such as NO.6~9 SEQ ID
Shown in any.
4. a kind of expression vector according to claim 3, which is characterized in that the skeleton of the expression vector includes existing
The expression vector of Escherichia coli.
5. a kind of expression system, which is characterized in that containing nucleotide sequence as shown at least one in NO.6~9 SEQ ID
Control the element of gene expression.
6. a kind of element for controlling gene expression described in claim 1 is in the application of Metabolism of E. coli engineering field.
7. the element of the control gene expression of one group of gradient intensity as claimed in claim 2 is in Metabolism of E. coli engineering field
Using.
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Non-Patent Citations (4)
| Title |
|---|
| Obtaining a Panel of Cascade Promoter-5′-UTR Complexes in Escherichia coli.;Zhou Shenghu ,等;《ACS Synth. Biol》;20170302;第6卷;第1065-1075页 * |
| 一种带增强子的原核高效表达载体的构建及初步应用;罗文新 等;《生物工程学报》;20000930;第16卷(第5期);第578-581页 * |
| 人工合成启动子文库研究进展;余君涵， 等;《微生物学报》;20160120;第43卷(第1期);第198-204页 * |
| 短短小芽孢杆菌-大肠杆菌穿梭分泌表达载体的构建;彭清忠 等;《生物工程学报》;20020731;第18卷(第4期);第438-441页 * |
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