CN106591310A - Escherichia coli high-strength combined promoters and application thereof - Google Patents
Escherichia coli high-strength combined promoters and application thereof Download PDFInfo
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Abstract
The invention discloses escherichia coli high-intensity combined promoters and an application thereof, and belongs to the field of biosynthesis technology. According to the invention, high-transcription-intensity promoters, namely PssrA, PdnaKJ, PgrpE and PalsRBACE, as well as a high-translation-intensity promoter, namely PrpsT, are provided; and the PssrA, PdnaKJ, PgrpE and PalsRBACE and PrpsT are fused, so that four combined promoters, which are high in expression intensity, are obtained. The maximum intensity of the combined promoter is 1.7 times as much as a 10mM arabinose induced PBAD promoter. The combined promoter is a safe and effective gene expression element.
Description
Technical field
The present invention relates to a kind of escherichia coli high-strength combination promoter and its application, belong to synthesising biological technical field.
Background technology
As first control element of gene expression, the table of gene downstream is controlled by the intensity of regulation and control promoter
Become most direct effectively means up to level.During metabolic engineering, the strategy such as modularity is using 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.
At present, a series of inducible promoter is most commonly used that in escherichia coli, yet with inducible promoter
Quantity is limited, therefore these promoteres are often difficult to carry out accuracy controlling to each Gene expression intensities.Microorganism
Substantial amounts of natural promoter is carried on genome, these promoter intensity are various, with being applied to the huge of metabolic engineering
Big potentiality.However, as the intensity of most of constitutive promoter is unknown, and promoter precise sequence corresponding with intensity is not
Know so that directly to carry out gene expression using constitutive promoter difficult.In addition the transcription of most natural promoter is strong
The translation intensity disunity in gene is spent, often the protein of the translation of the promoter with high transcriptional level is not but high, and low
The promoter of transcriptional level but possesses higher protein expression level.Often multiple promoteres, enhancer and decline on genome
Subtract the expression that the elements such as son jointly adjust a gene, therefore the promoter region of different length its function is also not quite similar.Such as
What finds the constitutive promoter with high Intensity of Transcription of Endothelial and high translation intensity becomes key.
The content of the invention
In order to solve the above problems, present invention firstly provides one group of escherichia coli constitutive promoter, clearly determines
Its Intensity of Transcription of Endothelial and translation intensity, there is provided its clear and definite DNA sequence.The present invention is to avoid the derivant during gene expression
Addition is there is provided selection, while can be used to adjust polygenic expression by way of control bacterial metabolism flow direction.
The nucleotide sequence of the escherichia coli constitutive promoter is as shown in SEQ ID NO.6～9 are arbitrary.
The present invention also provides a kind of expression vector, with startup of the nucleotide sequence as shown in SEQ ID NO.6～9 are arbitrary
Son.The skeleton of the expression vector includes common colibacillary expression vector.
The present invention provides the recombinant bacterium built using the promoter.
In one embodiment of the invention, the host of the recombinant bacterium includes various existing escherichia coli expression places
It is main.
In one embodiment of the invention, the recombinant bacterium for building using the promoter, is using described startup
Son connection genes of interest, construction recombination plasmid, and express in host.
In traditional genetic engineering research, inducible promoter is frequently used to express related gene, yet with need
Derivant to be added and it is difficult to while being finely adjusted to multiple Gene expression intensities, inducible promoter is dfficult to apply to many
Gene Expression Pathway.Constitutive promoter from genome is often due to the relatively low satisfaction that is equally difficult to of its expression intensity will
Ask.4 kinds of high-strength combination promoteres with gradient intensity that the present invention is provided do not need extra addition derivant, possess height
Intensity of Transcription of Endothelial and high translation intensity, at the same its that there is certain intensity gradient (promoter intensity span is 7.7 times) can be used for is right
Multi-gene expression approach is finely adjusted.
Specific embodiment
Materials and methods
E. coli jm109 is used for plasmid construction, and it is strong that e. coli k12 MG1655 is used for expressing protein measure promoter
Degree.
Embodiment 1
Analysis escherichia coli transcript profile data, with e. coli k12 MG1655 genomes as masterplate, expand promoter
PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsT.To determine the intensity of this 5 promoteres, merge P in the way of fusion DNA vaccinessrA、
PdnaKJ、PgrpE、PalsRBACE、PrpsTWith reporter gene EGFP.
To determine and building high-strength combination promoter, by promoter P in the way of fusion DNA vaccinessrA、PdnaKJ、PgrpEWith
PalsRBACETranscriptional control regions (- 10th area and -35th area) and promoter PrpsT5'-UTR regions fusion, obtain 4 so as to build
Plant combination promoter PssrA-rpsT、PdnaKJ-rpsT、PgrpE-rpsTAnd PalsRBACE-rpsT.Subsequently will combination promoter and EGFP gene with
The mode of fusion DNA vaccine merges for determining combination promoter intensity.Pcr amplification reaction is by high-fidelity DNA polymerase
PrimeSTAR HS (premix) (TaKaRa) are expanded.Amplimer is shown in Table 1.
1 the primer sequence of table
Artificial sequence
Fusion DNA vaccine product is connected with the pCDFDuet-1 plasmids of same double digestion after BamHI/SacI double digestions,
In chemical conversion e. coli k12 MG1655.Correct bacterial strain is sequenced for follow-up promoter strength detection.
1st, Intensity of Transcription of Endothelial
Recombinant bacterium 37 DEG C in LB culture medium or MOPS, under the conditions of 220rpm cultivate, cultivate to stable phase (11h) after
4000rpm, 4 DEG C are collected by centrifugation thalline, extract total mRNA of thalline, and real-time quantitative PCR detection is carried out after reverse transcription, determine EGFP
Intracellular mRNA abundance, testing result shows, using promoter PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsT, its mRNA abundance
It is the P of 10mM arabinose induction respectivelyBAD46.3,25.6,8.3,6.4 and 0.0047 times of promoter.
2nd, translate intensity
In order to determine constitutive promoter PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsTTranslation intensity, with EGFP as table
Up to gene, the stable phase detection intracellular detection intracellular EGFP level in escherichia coli MG1655.Recombinant bacterium is cultivated in LB or MOPS
37 DEG C in base, cultivate under the conditions of 220rpm, cultivate to stable phase (11h) after 4000rpm, 4 DEG C are collected by centrifugation thalline, utilize
PBS (pH=7.4) buffer solution for cleaning cell 2 times, to remove dead thalline, thalline secretions and culture medium precipitate component so as to reduce
Fluoroscopic examination background.By the thalline after cleaning as in 96 hole fluorescent screens in Multifunction fluorescent microplate reader fluorescence intensity and bacterium
Body OD600, testing conditions are excited for 488nm, 520nm transmittings.Promoter translation intensity is fluorescence intensity and OD600Ratio.
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.
Contrast promoter PssrA、PdnaKJ、PgrpE、PalsRBACEAnd PrpsTThe transcription and translation intensity of EGFP is found, is started
Sub- PssrA、PdnaKJ、PgrpEAnd PalsRBACEWith higher Intensity of Transcription of Endothelial, and it is relatively low to translate intensity.Promoter PrpsTHave
Relatively low Intensity of Transcription of Endothelial, and it is higher to translate intensity.Analysis reason, it may be possible to due to promoter PssrA、PdnaKJ、PgrpEAnd PalsRBACE
Promoter P containing strong transcriptional control regions (- 10th area and -35th area)rpsTThen there is stronger 5'-UTR regions.Therefore combine
Both sides' advantage is possible to build the combination promoter for obtaining high intensity
3rd, combine the translation intensity of promoter
In order to determine combination promoter P after promoter is engineeredssrA-rpsT、PdnaKJ-rpsT、PgrpE-rpsTWith
PalsRBACE-rpsTExpression intensity, with EGFP as expressing gene, in escherichia coli MG1655 stable phase detection intracellular detection born of the same parents
Interior EGFP levels.Fluorescence microplate reader testing result shows that the translation intensity of combination promoter is respectively the induction of 10mM arabinose
PBAD1.70,1.37,0.80 and 0.22 times of promoter.It follows that the four kinds of combination promoteres obtained through 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.
4th, combine the universality of promoter expressing gene
As four kinds of combination promoteres are respectively provided with identical 5'-UTR region, therefore, in order to detect that this combined serial starts
Sub- 5'-mRNA can only choose promoter P for heterogeneic ability to expressssrA-rpsTThe other reporter genes of expression, and
Its expression intensity is determined, so that it is determined that deriving from PrpsT5'-mRNA impacts that different genes are expressed.
P is used respectivelyssrA-rpsTCombination promoter and red fluorescent protein (RFP) and beta galactosidase (LacZ) fusion table
Reach, subsequently determine its translation intensity and be respectively PBAD1.4 times of promoter and 1.7 times.It is demonstrated experimentally that for different purpose bases
Cause, combination promoter equally have higher expression intensity.
5th, combine the negative sense result that promoter builds
It should be noted that due to the impact of mRNA secondary structures, and promoter original can be destroyed when sub-portfolio is transformed to starting
Some functional areas, therefore the transcript regions and 5'-UTR regions of combination high intensity promoter might not obtain forward junction
Really.
Using another kind of high translation intensity constitutive promoter PinfC-rplT5'-UTR regions and PssrA、PdnaKJ、PgrpE、
PalsRBACETranscript regions combination build 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 promoteres：Its translation intensity difference
For PinfC-rplT0.94,0.58,0.51,0.21 times.This several combination promoter intensity has declined.
Although the present invention is disclosed as above with preferred embodiment, which is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be by being defined that claims are defined.
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 (8)
1. it is a kind of control gene expression element, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO.6～9 are arbitrary.
2. a kind of gradient intensity control gene expression element, it is characterised in that including nucleotide sequence such as SEQ ID NO.6
At least 2 in promoter shown in～9.
3. a kind of expression vector, it is characterised in that the nucleotide sequence of its gene expression control elements such as SEQ ID NO.6～9
Shown in arbitrary.
4. a kind of expression vector according to claim 3, it is characterised in that the skeleton of the expression vector includes existing
Colibacillary expression vector.
5. a kind of expression system, it is characterised in that containing nucleotide sequence as shown at least one in SEQ ID NO.6～9
The element of control gene expression.
6. a kind of expression system according to claim 5, it is characterised in that the host of the recombinant bacterium is escherichia coli.
7. application of the element of a kind of control gene expression described in claim 1 in metabolic engineering field.
8. application of the element of the control gene expression of a kind of gradient intensity described in claim 1 in metabolic engineering field.
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| CN201611126585.7A CN106591310B (en) | 2016-12-09 | 2016-12-09 | A kind of Escherichia coli high-strength combination promoter and its application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116875594A (en) * | 2023-04-26 | 2023-10-13 | 江南大学 | Construction and application of pediococcus acidilactici high-strength combined promoter |
-
2016
- 2016-12-09 CN CN201611126585.7A patent/CN106591310B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| ZHOU SHENGHU ,等: "Obtaining a Panel of Cascade Promoter-5′-UTR Complexes in Escherichia coli.", 《ACS SYNTH. BIOL》 * |
| 余君涵， 等: "人工合成启动子文库研究进展", 《微生物学报》 * |
| 彭清忠 等: "短短小芽孢杆菌-大肠杆菌穿梭分泌表达载体的构建", 《生物工程学报》 * |
| 罗文新 等: "一种带增强子的原核高效表达载体的构建及初步应用", 《生物工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116875594A (en) * | 2023-04-26 | 2023-10-13 | 江南大学 | Construction and application of pediococcus acidilactici high-strength combined promoter |
| CN116875594B (en) * | 2023-04-26 | 2024-01-30 | 江南大学 | Construction and application of pediococcus acidilactici high-strength combined promoter |
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| Publication number | Publication date |
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| CN106591310B (en) | 2019-10-25 |
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