CN110029121A - A kind of construction method of pseudomonas aeruginosa CRISPRi system - Google Patents
A kind of construction method of pseudomonas aeruginosa CRISPRi system Download PDFInfo
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- CN110029121A CN110029121A CN201910307818.0A CN201910307818A CN110029121A CN 110029121 A CN110029121 A CN 110029121A CN 201910307818 A CN201910307818 A CN 201910307818A CN 110029121 A CN110029121 A CN 110029121A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Abstract
The invention discloses a kind of construction methods of pseudomonas aeruginosa CRISPRi system, this method includes one derivable shuttle plasmid of selection, CRISPRi-dCas9 gene is inserted into shuttle plasmid, indispensable gene and dispensable gene according to pseudomonas aeruginosa, corresponding sgRNA expressed sequence is designed, finally sgRNA expressed sequence is inserted into above-mentioned recombinant plasmid up to CRISPRi system.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of building side of pseudomonas aeruginosa CRISPRi system
Method.
Background technique
Pseudomonas aeruginosa is the important pathogen pseudomonas aeruginosa of infection of burn, is burning, trauma patient local infection
With the encountered pathogenic bacteria of general infection.The data that investigation in net 2016 is announced, P. aeruginosa are detected according to Chinese bacterial resistance
5 are arranged in bacterium separation pathogen all in clinic, account for 8.66%.In Burn Ward, pseudomonas aeruginosa is all
Composition ratio in pathogen is higher, reaches 18-43.6%.In many Burn Wards, especially the burn ward ICU, verdigris is false
Monad replaces with Acinetobacter bauamnnii is in the highest pathogenic bacteria of recall rate.Pseudomonas aeruginosa compares Acinetobacter bauamnnii
Carry more virulence factors.Other than pod membrane, endotoxin, pseudomonas aeruginosa also carries type III excretory system, Ke Yifen
Secrete the virulence proteins such as ExoU and ExoS.Pseudomonas aeruginosa can cause local infection, including skin, subcutaneous tissue, bone, ear,
Eye, urinary tract, heart valve and the infection of art area, can also cause systemic infection.Charrin disease not only results in burn
Wounds are deepened, and skin-grafting failure can also cause burn general infection and septic shock, be the important disease for leading to death
Opportunistic pathogen.It makes the matter worse, the drug resistance of pseudomonas aeruginosa is also increasingly serious.Chinese bacterial resistance detects 2016 annual data of net
It points out, is 28.7% to the resistant rate of Imipenem in the pseudomonas aeruginosa of China's separation.Through investigating, it was also found that burning
The drug resistance of ward pseudomonas aeruginosa is more acute, has all reached 65% or more to the resistant rate of Imipenem and Meropenem.
The recall rate of multi-resistant Pseudomonas aeruginosa and general tolerant Pseudomonas aeruginosa is also very high simultaneously.Multi-drug resistant bacteria
The separation rate of (Multidrug-resistant, MDR) clinically is 54.59%, and general antibody-resistant bacterium (Extensively
Drug Resistant, XDR) separation rate in clinic reaches 21.42%.General tolerant Pseudomonas aeruginosa refer to only to 1~
2 kinds potential have the active drug of anti-acinetobacter calcoaceticus [refer mainly to polymyxin) sensitive bacterial strain.General tolerant Pseudomonas aeruginosa
Occur seriously threatening the life security of fire victim, has beaten alarm bell to the mankind.
The antibiosis of current clinically available charrin disease is known as beta-lactam class, Carbapenems, amino
Glycoside, quinolones and colistin (CONTINENTAL AREA OF CHINA is unavailable) etc..The target spot of the above antibiotic concentrates on penicillin knot
The indispensable gene of the bacteriums such as hop protein, 30S subunit 16S rRNA and DNA gyrase.The indispensable gene of bacterium is exploitation antibiotic
Important target spot, the antibiotic clinically used at present is all targeted bacteria indispensable gene.Therefore, it needs false to verdigris single
The indispensable gene of born of the same parents bacterium is furtherd investigate, to find more potential antibiotic target spots.
The indispensable gene quantity of document report pseudomonas aeruginosa is a or so in 300-400 at present, different bacterial strains, or
Different condition of culture slightly has difference with the quantity that different methods obtains indispensable gene.Although pseudomonas aeruginosa is required
Number of genes and type are substantially clear, but the function of many indispensable genes still lacks research, in pseudomonas aeruginosa PAO1
In have 46 indispensable gene Unknown Functions.The function of pseudomonas aeruginosa indispensable gene is furtherd investigate, can help to seek
New antibiotic action target spot is looked for, increasingly serious resistance problems are coped with.The functional study method of traditional bacterium indispensable gene
Include: 1, RNA interfering (RNAi) regulates and controls the expression of indispensable gene by siRNA.2, pass through the method for replacement, present gene
It is derivable mutually homogenic to be inserted into one for other positions in group, then knocks out the gene put in situ.The advantages of both the above method, exists
In technology maturation, disadvantage is operating process complexity, is not readily used for high pass quantity research. CRISPR interference
(CRISPRi) technology is in recent years for studying an efficiently strong means of gene function.
CRISPR-Cas9 is current most widely used CRISPR system, in addition to be used for cell line, stem cell and
Outside the gene for inducing the editor's prokaryotic cell and eukaryocyte in versatile stem cell level rapidly and efficiently, also a lot of other
Field is widely used.Draw for example, specificity is used in combination using the CRISPR-Cas9 albumen (abbreviation dCas9 albumen) of inactivation
RNA is led, can achieve the purpose of a certain gene expression dose of specific regulatory control.Currently, the library sgRNA of high overlay capacity is absurd fantastic
It is raw, each gene of cell can be covered, then by the method for positive-negative selection, screen interested access or gene,
This technology has expanded the method that scientist studies functional genomics significantly.Target spots of many viruses or virulence factor and again
Signal path is wanted to be identified by this method.It is equally to make it under sgRNA guidance using the Cas9 technology of inactivation
In conjunction with bacterium indispensable gene to block its transcription, without cutting off DNA, the table of certain indispensable genes can be inhibited in this way
It reaches, some indispensable genes is studied in bacteria levels.
CRISPR-Cas is the acquired immune system of bacterium, passes through specific recognition and shears bacteriophage or plasmid
Particular sequence and then the infection for removing Exogenous Nucleic Acid.CRISPR-Cas system is capable of the identification of specificity and shears double-strand
DNA, this characteristic are exploited for the gene editing of bacterium and cell.CRISPR-Cas system be present in 45% bacterium and
In 87% archeobacteria.Different according to the number of cas albumen, CRISPR-Cas can be divided into 2 major class i.e. class l and at present
2 class of class.CRISPR-Cas9 system belongs to the 2nd class II type CRISPR-Cas system, is currently used for eukaryocyte and original
The gene editing of nucleus is most widely used, most representative CRISPR-Cas system.CRISPR-Cas9 system in addition to
In gene editing, gene expression regulation can be also used for, such as CRISPR activation (CRISPRa) and CRISPR-Cas9
interference(CRISPRi).The principle is as follows: Cas9 protein cleavage double-stranded DNA relies on two nuclease domains: HNH
Domain and the domain RuvC.Each amino acid sites (D10A and H841A) in HNH structural domain and the domain RuvC are mutated, so that mutation
Cas9 albumen afterwards loses the ability of shearing DNA, i.e. dCas9 (nuclease-deactivated Cas9), although dCas9
The ability of shearing DNA is lost, but still is able to be integrated to target sequence under the guidance of sgRNA.
Summary of the invention
The present invention provides a kind of construction method of pseudomonas aeruginosa CRISPRi system and is applied to inquire into, and wraps
Include following 4 steps:
1) selection and the derivable shuttle plasmid PHERDB20 of digestion, by dCas9 (abbreviation of CRISPR-dCas9, below
Gene is inserted into the Arabinose promoter downstream (method of digestion connection) in PHERDB20 shuttle plasmid herein all together)；It is first
First successfully construct PHERDB20-dCas9 recombinant plasmid
2) according to the indispensable gene and/or dispensable gene of document report pseudomonas aeruginosa, based on special moral principle, choosing
Suitable sgRNA sequence is taken, is inserted into above-mentioned recombinant plasmid PHERDB20-dCas9 by the method for Ji Busen clone, building
PHERDB20-dCas9-sgRNA recombinant plasmid.
Alternative: the expressed sequence (promoter+20nt target sequence+handle structure+termination of sgRNA is directly synthesized
Son), pass through the method for digestion (EcoRI/KpnI or XbaI/SalI restriction enzyme site is designed at both ends when synthesis) connection, building
PHERDB20-dCas9-sgRNA recombinant plasmid, as CRISPRi system.
3) different indispensable genes is chosen, the CRISPRi system of building is verified.
4) the potential antibiotic action target spot of CRISPRi screening system based on building
The method of aforementioned present invention targets sequence choosing sgRNA, is based on three cardinal principles: a) expressed sequence
Normal chain on have the sequence of CCN, b) sequence length of the expressed sequence is about 20nt, and close to the area ATG (transcription initiation
Codon) and c) sequence of the expressed sequence has specificity.
The method of aforementioned present invention, the pseudomonas aeruginosa are preferably PAO1 bacterial strain, what the PAO1 bacterial strain had confirmed that
Essential gene at least 352, the indispensable gene of the known function, representativeness is prtR, the core of corresponding sgRNA
Nucleotide sequence is as shown in SEQ ID NO.1, and bp14-16 nucleotide are PAM sequences in sequence, and bp17-36 nucleosides
Acid is target sequence.
The method of aforementioned present invention passes through at least 352 pairs of gRNA aligning primers of the essential gene of PAO1 bacterial strain
Gibson assembling, is cloned into respectively in pHERD20T-dCas9 plasmid, constructs the library CRISPRi.The CRISPRi of the building
Library is pHERD20T-dCas9-gRNA001 to pHERD20T-dCas9-gRNA352.
The method of aforementioned present invention, the indispensable gene of the unknown function, representativeness is PA0715, corresponding
The nucleotide sequence of sgRNA is as shown in SEQ ID NO.2, and bp13-15 nucleotide is PAM sequence in sequence, and bp16-
40 nucleotide are target sequence.
In one embodiment, the side of the invention that CRISPR-dCas9 system is constructed in pseudomonas aeruginosa
The gRNA of above-mentioned design is inserted into CRISPR-dCas9 system by method, constructs the library CRISPRi, the verifying library CRISPRi
Efficiency.The following steps are included:
1) using pHERD20T shuttle plasmid as skeleton, by the Arabinose promoter of dCas9 gene cloning to pHERD20T
Downstream constructs pHERD20T-dCas9, and detailed process is as follows:
A) using plasmid in AddGene as template, design p-dCas9 or more primer pair, PCR amplification dCas9 gene, purifying,
DNA fragmentation is recycled,
The DNA genetic fragment of dCas9 is connected to (arabinose in pHERD20T plasmid by b) digestion pHERD20T plasmid
At the multiple cloning sites in promoter downstream), resistant panel (ammonia benzyl) screens recon, constructs pHERD20T-dCas9, and sequencing is tested
Card；
2) it by taking representative prtR gene (inhibiting pyocin secretor) as an example, identifies in pseudomonas aeruginosa
The inhibition efficiency of CRISPRi, detailed process is as follows:
A) according to the sequence of fixed prtR, sequence is targeted based on the gRNA that " three principles " chooses prtR, design is complementary
Primer P-prtR-up and P-prtR-down, b) method that is assembled by Gibson, the gRNA targeting sequence of prtR is inserted into
In pHERD20T-dCas9 plasmid, recon is screened, constructs pHERD20T-dCas9-prtR, sequence verification；
3) the corresponding 352 pairs of gRNA aligning primers of 352 essential genes are synthesized, is assembled, is cloned by Gibson
In pHERD20T-dCas9 plasmid, the library CRISPRi is constructed i.e.: pHERD20T-dCas9-gRNA001 to pHERD20T-
dCas9-gRNA352.Each pHERD20T-dCas9-gRNA carries out sequence verification.
In another embodiment, method of the invention, comprising: 1. directly by the insertion PAO1 dyeing of dCas9 gene
In body, building PAOl-dCas9 gene knock-in bacterial strain (dCas9 gene can arabinose inducing expression)；2. by 352 pairs of gRNA sequences
Column are inserted into shuttle plasmid Pucp24.3. recombinant plasmid Pucp24-gRNA352 is transferred in PAO1-dCas9 bacterial strain.
Potential antibiotic work is being screened in the pseudomonas aeruginosa CRISPRi system of method building of the invention or library
With the purposes of target spot, the antibiotic is the antibiotic for treating pseudomonas aeruginosa and green pseudomonad CRISPRi system
For studying the purposes of the indispensable gene function of pseudomonas aeruginosa, and regulating and controlling and inhibiting the required of pseudomonas aeruginosa
The purposes of gene expression.
The process of CRISPRi system or library screening antibiotic action target spot of the invention is as follows:
With method of the invention, corresponding sgRNA is designed according to the indispensable gene of pseudomonas aeruginosa PAO1 and expresses sequence
Column, each sgRNA sequence is cloned into CRISPR-dCAS9 system, establishes the library CRISPRi (the i.e. verdigris vacation of indispensable gene
The set of each indispensable gene CRISPRi system of monad).The efficiency that CRISPRi system inhibits indispensable gene expression is verified, it is right
Indispensable gene carries out the research of high-energy phenotype, morphologic detection: such as bacterium colony, growth, power, biomembrane parameter, from filtering out
Antibiotic action target spot.Specific screening process is shown in Fig. 7.
CRISPRi system of the invention for study bacterium indispensable gene have several advantages that it is 1) easy to operate,
The sequence for only needing to design sgRNA can be constructed within 1 week by common molecular clone and complete a target gene
CRISPRi system；2) high pass quantity research can be carried out, by designing the sgRNA sequence of different genes, multiple genes can be carried out
Regulation；3) high-efficient, it can induce control.
Detailed description of the invention
Fig. 1 CRISPR-dCas9 system is successfully transferred to the electrophoretogram in pseudomonas aeruginosa PAO1；
Fig. 2 CRISPR-dCas9 system is successfully transferred to the electrophoretogram in pseudomonas aeruginosa PAK；
The inhibition of Fig. 3 indispensable gene PA0715 can promote the secretion of PAO1 pyo；
Fig. 4 various concentration induces influence of the CRISPR-dCas9-sgRNAprtR to PAO1 bacterial growth.
Specific embodiment
Following embodiment introduces CRISPRi system using an indispensable gene prtR of pseudomonas aeruginosa PAO1 as representative
With the specific construction method and verification method in the library CRISPRi.But it does not limit CRISPRi system of the invention with this only to limit
In prtR gene, the building of the other indispensable genes of pseudomonas aeruginosa or dispensable gene CRISPRi system is extended to.
Reagent and material
The building of embodiment 1CRISPRi system
1, selection and processing shuttle plasmid
By designing and screening, select shuttle plasmid PHERDB20T as expression vector.
PHERDB20T is that University Medical College molecular genetic and microbiology teaching and research room Schweizer religion are learned by Florida
It awards and gives, which is amicillin resistance, has the ability replicated in Escherichia coli and pseudomonas aeruginosa, simultaneously
Promoter with the induction of an arabinose, and the sequence on plasmid also with one section of plasmid engagement transfer identification.
2, PCR amplification dCas9 gene
1) primer is as follows:
pdCas9-bsaI-R:(EcoRI)cgcGAATTCgagaccTTAGTCACCTCCTAGCTGAC
pdCas9-bsaI-F:(EcoRI)tccggtctcGAATTCGATGGATAAGAAATACTCAAT
Template is pdCas9-bacteria (Addgene number is 44249)
2) PCR amplification dCas9 gene, using pdCas9-bacteria as template, pdCas9-bsaI-R and pdCas9-
BsaI-F is primer, expands dCas9 gene, and reaction condition is as follows:
3) PCR reaction condition:
Step1:95 DEG C of initial denaturation 5min.
Step2:95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s are recycled 30 times.
Step3:72 DEG C of extension 10min.
4) PCR product glue recycles: prepare 1% agarose gel, 50ul whole loading, and 60V electrophoresis 60 minutes, according to
Marker cuts required band with blade, recycles DNA with the plastic recovery kit of Promega.Substantially steps are as follows: root
According to the glue under cutting off weight (1mg corresponds to 3ul DNA binding buffer) be added DNA binding buffer, 60 DEG C
It is incubated for 10 minutes, can suitably mix twice to accelerate peptization solution.Glue lysate containing DNA is transferred in DNA adsorption column, from
It is washed twice after the heart with wash buffer, finally uses the ddH of 20ul2O eluted dna.
5) digestion and recycling of dCas9
Endonuclease reaction condition:
0.8% agarose gel is prepared, 15ul whole loading 60V electrophoresis 60 minutes, according to marker, is cut with blade
Required band recycles DNA with the plastic recovery kit of Promega.Substantially steps are as follows: according to the weight of the glue under cutting off
It measures (1mg corresponds to 3ul DNA binding buffer) and DNA binding buffer is added, 60 DEG C are incubated for 10 minutes, can be appropriate
It mixes twice to accelerate peptization solution.Glue lysate containing DNA is transferred in DNA adsorption column, and wash buffer is used after centrifugation
It washes twice, finally with the deionized water eluted dna of 6ul to get dCas9 gene.
3, the connection of plasmid PHERDB20T and dCas9
By verifying correct plasmid, for connecting dCas9 segment in next step, since dCas9 segment both ends are to use BsaI
The EcoRI cohesive end of identification, both ends are EcoRI cohesive ends, therefore are needing further to select direction of insertion just after connecting
True plasmid.
1) single endonuclease digestion of PHERDB20T
37 DEG C digestion 3 hours, 1% agarose gel electrophoresis, recycle purpose band segment.
3) connection of PHERDB20T and dCas9
DCas9 viscosity segment is to be cut out after front PCR amplification obtains with BsaI is mono-, can directly be taken out from -20 DEG C
It connects
The connection of PHERDB20T and dCas9
Reaction condition:
16 DEG C of connections are overnight.
4, the building of PHERDB20-dCas9 recombinant plasmid
1) connection product is converted into Escherichia coli.Connection liquid 5-10ul is taken, 50ul e. coli bl21 is respectively added
(DE3) competent cell is incubated for 30 minutes on ice, heat shock 90 seconds, ice bath 2 minutes, the LB of 1ml preheating, 37 DEG C of recoveries 1 is added
A hour applies the plate of ampicillin.37 DEG C of overnight incubations.
2) PCR amplification
Single colonie on picking plate with universal primer carry out colony PCR amplification (primer: pHerDB20T-F:
GATGGAGTGAAACGATGGCG；pHerDB20T-R:ACGTTGTAAAACGACGGCCA).
Amplification condition:
PCR reaction condition:
Step1:95 DEG C of initial denaturation 5min.
Step2:95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 240s are recycled 30 times.
Step3:72 DEG C of extension 10min.
Prepare 2% agarose gel, electrophoresis detection.
3) it chooses positive bacterium colony to be inoculated in the LB culture medium of 3ml, 37 DEG C of overnight incubations.Next day extracts plasmid, specific to walk
Suddenly see Promega plasmid extraction kit, finally use 50ulddH2O elution, obtains.
4) plasmid order-checking is verified, and obtains PHERDB20-dCas9 recombinant plasmid.
5, the building of PHERDB20-dCas9-sgRNA recombinant plasmid (CRISPRi system)
1) sequence of design synthesis sgRNA.According to pseudomonas aeruginosa PAO1 known function indispensable gene prtR
With the indispensable gene PA0715 of 1 one unknown functions, sequence (the prtR-sgRNA such as SEQ ID of corresponding sgRNA is designed
Shown in NO.1 and PA0715-sgRNA such as SEQ ID NO.2), gene chemical synthesis sgRNA gene plasmid by conventional method, both ends
Design restriction enzyme site EcoRI/KpnI, XbaI/SalI.
2) double digestion obtains sgRNA segment
The sgRNA gene plasmid for taking design to synthesize does digestion, uses EcoRI/KpnI, XbaI/SalI to do double digestion respectively,
The sgRNA segment for cutting out prtR and PA0715, cuts glue, in case subsequent and dCas9 (CRISPRi-dCas9) is connected to wears together
On shuttle plasmid.
The endonuclease reaction condition for obtaining the sgRNA segment of prtR is as follows:
37 DEG C digestion 3 hours, 1.5% agarose gel electrophoresis.
The endonuclease reaction condition for obtaining PA0715-sgRNA segment is as follows:
37 DEG C digestion 3 hours, 1.5% agarose gel electrophoresis.
3) glue recycles target DNA fragment: the sgRNA segment of prtR and PA0715 that upper step digestion is gone out are recycled using glue
Kit is cut the DNA band of 150bp or so, is recycled, steps are as follows:
A takes an EP pipe, marks title, weigh weight；
B after low voltage electrophoresis, cuts the DNA fragmentation of purpose size strip, rinses every time or more allowing blade replacement is to avoid DNA dirt
Dye.Glue containing DNA is put into EP pipe, is weighed again；
C calculates the net weight of glue, and according to the ratio of 100mg:100ul, DNA binding soln, 55 DEG C of -60 DEG C of dissolutions 10 are added
Minute, it thoroughly dissolves and (can be vibrated in course of dissolution twice to accelerate dissolution, ensure that glue has been completely dissolved before upper prop) to glue；
Glue lysate is transferred in DNA adsorption column by D, and 10000g, which is centrifuged 30 seconds, (can be added again DNA for glue lysate
It is centrifuged in adsorption column)；
E takes the wash buffer of 200ul to wash DNA column twice, after last time 13000g is centrifuged 2 minutes, takes out
DNA column room temperature is volatilized 5 minutes (allowing ethyl alcohol thoroughly to volatilize, to lower the influence to downstream experiment)；
F, the final deionized water eluted dna for using 10ul take 1ul electrophoresis detection concentration, and -20 DEG C of preservations are spare.
It recycles to obtain prtR-sgRNA and PA0715-sgRNA by the above method.
4) connection of plasmid PHERDB20-dCas9 and sgRNA
1) reaction condition is connected:
16 DEG C of connections overnight, obtain PHERDB20-dCas9-sgRNA connection product.
Connection product is converted into Escherichia coli
2) connection liquid 5-10ul is taken, 50ul e. coli bl21 (DE3) competent cell is respectively added, is incubated for 30 on ice
Minute, heat shock 90 seconds, ice bath 2 minutes, the LB of 1ml preheating is added, 37 DEG C of 1 hours of recovery, applies the plate of ampicillin.
37 DEG C of overnight incubations.
3) PCR amplification, plasmid order-checking verifying, obtaining PHERDB20-dCas9-sgRNA recombinant plasmid connection product is
Pseudomonas aeruginosa CRISPRi system, PHERDB20T-dCas9-PA0715 or PHERDB20T-dCas9-PrtR, can be with
Targeted inhibition PA0715 and PrtR indispensable gene respectively.
The building in 2 library pseudomonas aeruginosa CRISPRi of embodiment
Referring to the method for embodiment 1, the corresponding 352 pairs of gRNA aligning primers of 352 essential genes of PAO1 bacterial strain are synthesized,
It is assembled, is cloned into pHERD20T-dCas9 plasmid by Gibson, be built into the essential gene that 352 have confirmed that
CRISPRi system, these CRISPRi system sets be the library CRISPRi i.e.: pHERD20T-dCas9-gRNA001 is extremely
pHERD20T-dCas9-gRNA352.Each
PHERD20T-dCas9-gRNA carries out sequence verification.
CRISPRi system and library of the embodiment 3 using other indispensable genes of the building pseudomonas aeruginosa of embodiment 2,
Functional study is carried out to the indispensable gene of pseudomonas aeruginosa PAO1 by the system or library.
1, it looks first at, PHERDB20T-dCas9-prtR and PHERDB20T-dCas9-PA0715 are to P. aeruginosa
The influence of bacterium growth conditions
1.1 plasmid PHERDB20T-dCas9-prtR-sgRNA and PHERDB20T-dCas9-PA0715-sgRNA
It is transferred to pseudomonas aeruginosa
1) trilinear method recovery PAO1 and PAK bacterium from liquid nitrogen, 37 DEG C of overnight incubations；
2) the picking single colonie from plate, the overnight incubation in LB liquid medium；
3) next day takes 1.5ml to be added in EP pipe, and 16000g is centrifuged 1min；
4) supernatant is removed, thallus is washed three times with the sucrose of 300mM；
5) after removing supernatant, thallus finally uses the sucrose further of the same concentration of 200ul, and every pipe dispenses 50ul；
6) electric shock cup is taken out from alcohol, is thoroughly dried up, and is irradiated several minutes under ultraviolet lamp, it is spare；
7) into 50ul competence, the Plasmid DNA of 1ul is added, pipette tips gently blow and beat mixing, are placed at room temperature for 2min；
8) it is transferred in electric shock cup, adjustment electroporation parameter, voltage 2.5kV, the common training of 1ml is rapidly added after electric shock
Base is supported, 37 DEG C are cultivated 1 hour；
9) 10 times of coubling dilutions calculate transformation efficiency, while 10ul being taken to be coated in ampicillin plate to succeed
Transformant.
1.2 plasmid PHERDB20T-dCas9-prtR and PHERDB20T-dCas9-PA0715 are raw to pseudomonas aeruginosa
The influence of long status
1) with method above-mentioned, by the PHERDB20T-dCas9-prtR of 1ul and PHERDB20T-dCas9-PA0715 electricity
It hits and is transferred in pseudomonas aeruginosa, the solution of 100ul is taken to apply carbenicillin plate；
2) next day picks from the plate transformant monoclonal, and being cultivated in LB liquid medium to OD600 is 1.0 or so；
3) bacterium solution of 100ul is taken, 10 times of dilution method dilutions take 3ul liquid to drip from dilution respectively and containing 0.02%
On the plate of arabinose and 1% glucose；
4) whether the bacterium amount that next day observation bacterium grows on different plates is consistent.
2. secondly, the recombinant plasmid that observation carries PHERDB20T-dCas9-prtR system converts pseudomonas aeruginosa
The influence of efficiency
Influence of the 2.1PHERDB20T-dCas9-prtR to pseudomonas aeruginosa PAO1 transformation efficiency
The result is shown in Figure 1, the result of Fig. 1 show that CRISPRi-Cas9 system is successfully transferred in pseudomonas aeruginosa PAO1.
As shown in Figure 4 A, under the induction of 0.02% arabinose, the recombinant plasmid of CRISPRi-Cas9 system is carried
(PHERDB20T-dCas9-prtR sgRNA) has significant impact to the transformation efficiency of bacterium, above a row be without containing
DCas9, the recombinant plasmid only containing sgRNA segment, below a row be the plasmid containing complete CRISPRi-Cas9 system.?
1% glucose is added on culture plate to inhibit the expression of Arabinose promoter, and then inhibits the background expression of dCas9.
It is added after glucose, the transformation efficiency of bacterium is again apparent to be restored.Compared with the only plasmid of sgRNA, transformation efficiency
Have no significant difference.
Influence of the 2.2PHERDB20T-dCas9-prtR to pseudomonas aeruginosa PAK transformation efficiency
The result of Fig. 2 shows that the recombinant plasmid electricity for carrying CRISPRi-Cas9 system is gone in PAK bacterial strain, same to send out
Existing PHERDB20T-dCas9-prtR sgRNA can significantly inhibit the transformation efficiency of PAK.Glucose can equally reverse this
Depression effect.
3.PA0715 inhibits that the secretion of PAO1 green pigment can be promoted
With method above-mentioned, we have selected the indispensable gene of a Unknown Function, PA0715 again.When PA0715 gene
Pseudomonas aeruginosa bacterium colony size, which has no, after being inhibited by CRISPRi substantially change, but under the induction of arabinose, bacterium
Equally there is obvious delay in growth in liquid medium, and performance is suppressed similar with prtR gene.Different places
It is, after PA0715 is suppressed, the secretion of green pigment (supposition is pyocyanin, is doing further verifying) is significantly raised
(see Fig. 3).
4 CRISPR-dCas9-sgRNAprtR various concentrations induce result
Further, various concentration arabinose is had detected to the induced efficiency of CRISPRi system.Shown in following Fig. 4, no
Influence with concentration arabinose induction CRISPR-dCas9-sgRNAprtR to PAO1 bacterial growth is different, generally presents
Concentration dependant trend.When arabinose concentrations are 0.1% and 0.05%, bacterium can not grow substantially.Work as arabinose concentrations
When lower than 0.025%, concentration is lower, and CRISPR-dCas9-sgRNAprtR is weaker to the inhibiting effect of PAO1.
The above results show the CRISPRi system established in pseudomonas aeruginosa be it is controllable, can be by adjusting luring
It leads agent concentration and realizes the different degrees of inhibition of target gene.
The present invention uses CRISPR-Cas9 technology, the expression of the prtR gene by inhibiting bacterium, thin to reach control
The purpose of bacterium.Compared with directly CRISPR system being used to remove gene, the advantages of this technology, is that bacterial genomes will not be made
At the change of science of heredity.In the research of early period, although using CRISPR system can efficiently bacteria removal, exist
One problem, even if it can make the quantity of bacterium reduce by 103The order of magnitude still has the bacterium of one thousandth or a ten thousandth logical
The method for repairing cut-off gene is crossed successfully to survive.For the bacterium of this small part mutation, drug resistance and virulence are
It is no to be substantially reduced not in-depth study.And inhibit the expression of a certain gene using CRISPR systemic characteristic, only
It adjusts at transcriptional level, is integrated to specific base jointly by the Cas9 albumen (i.e. dCas9 albumen) and sgRNA of inactivation
Because of region, the formation or extension of block either transcription complex and block its transcription.There is no the bases for shearing bacterium for this technology
Because of group, the probability of its mutation is not increased.But this technology with directly use CRISPR system remove gene compared with, target
The selection of gene often requires that higher.Gene directly, which is removed, using CRISPR system not only passes through drug resistant gene or virulence gene
It, can also be by any gene of targeted bacteria to achieve the purpose that bacteria removal etc. bacteria removal is achieved the purpose that.And it uses
CRISPRi (CRISPR interference) system needs non-target bacteria to grow required gene, by inhibiting these must base
Because of the expression of (essential gene), bacterium is reduced to certain quantity, then can be treated and be reached with combination antibiotic
The purpose of bacteria removal.The pressure and the probability for generating mutation that bacterium faces can be reduced in this way.
Above embodiments only enumerate representative pseudomonas aeruginosa indispensable gene PrtR and dispensable gene PA0715
The building of CRISPRi system and its to P. aeruginosa growth inhibitory effect test, according to embodiment 1 method construct copper
The CRISPRi system of green pseudomonad remaining indispensable gene and dispensable gene, builds up pseudomonas aeruginosa indispensable gene
The library CRISPRi.Make some simple changes under without departing from Spirit Essence of the invention and accommodation also belongs to model of the invention
It encloses.
Sequence table
<110>Affiliated Hospital of Zunyi Medical College
<120>a kind of construction method of pseudomonas aeruginosa CRISPRi system
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 771
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atggacaaga gcacccagat cccgcccgac agcttcgccg ctcgcctcaa gcaggccatg 60
gcgatgcgca acctgaagca ggaaaccctc gccgaagcgg caggggtttc gcagaacacc 120
attcacaagc tgacctcggg caaggcccag agcacccgca agctgatcga gatcgcggcg 180
gccctgggcg tctcgccggt ctggctgcag accggcgaag gcgctccagc cgcgcgcagt 240
gccgtgtccg tggccgatgg cagcccattg gtgctggaac cgctgcatcc gtgggacagc 300
gacacaccgc tggacgaaga cgaagtggaa ctgccgctgt acaaggaagt ggagatgtcc 360
gccggcgccg gacgcactgc ggtgcgcgag atagaggggc gcaagctgcg tttttcctac 420
gccacgctgc gtgcctcggg cgtcgatccg tcggcggcga tctgcgccca actcaccggc 480
aacagcatgg aaccgctgat catggatggc tccaccatcg gcgtggacac cgccaccacc 540
catatcaccg atggcgagat ctacgccctc gaacatgacg gcatgctgcg ggtgaagttc 600
gtctatcgcc tgcccggcgg cggcattcgc ctgcgcagct tcaaccgcga ggaatatccg 660
gacgaggagt actcgccgga ggacatgcgc agccgccaga tcagcatgat cggttgggtc 720
ttctggtggt ccaccgtacg ccaccggcgc ggcccgtccc tggtgcggtg a 771
<210> 2
<211> 200
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgaagaaga gacctttaga agatcttttt tttgcgatgt acagagggaa gcagagtttc 60
gaacactttg ccaattgctc tgtcgaaggt ttatatgagc cggtgatggt gaatggacga 120
ctcgtatata aaaccgataa ggatctgcgt gcgtatcatc ggtttctaaa taaattcttg 180
tttgaaaggc ttcctgttgt 200
Claims (10)
1. a kind of construction method of pseudomonas aeruginosa CRISPRi system, comprising the following steps:
1) dCas9 gene, is inserted by selection and the derivable shuttle plasmid PHERDB20 of digestion by digestion connection method
Arabinose promoter downstream in PHERDB20 shuttle plasmid constructs PHERDB20-dCas9 recombinant plasmid；
It 2) is every base based on special moral principle according to the indispensable gene and/or dispensable gene of document report pseudomonas aeruginosa
Because choosing suitable sgRNA sequence, pass through Ji Busen PCR cloning PCR inserting step 1) PHERDB20-dCas9 recombinant plasmid in, structure
PHERDB20-dCas9-sgRNA recombinant plasmid is built, or direct with promoter+20nt target sequence+handle structure+terminator
The expressed sequence for synthesizing sgRNA constructs PHERDB20-dCas9-sgRNA recombinant plasmid by digestion-connection method, completes
The building of CRISPRi system；
3) different indispensable genes is chosen, the CRISPRi system of building is verified, optional
4) the potential antibiotic action target spot of CRISPRi screening system based on building.
2. the method as described in claim 1, the sgRNA expressed sequence, it is characterised in that: a) expressed sequence just connects
On have the sequence of CCN, b) sequence length of the expressed sequence is about 20nt, and close to the area ATG (translation initiation codon),
And c) sequence of the expressed sequence has specificity.
3. the method as described in claim 1, the pseudomonas aeruginosa is PAO1 bacterial strain.
4. method as claimed in claim 3, the essential gene that PAO1 bacterial strain has confirmed that at least 352.
5. the method as described in claim 1, step 1) and 2) described in enzyme cutting be EcoRI/KpnI or XbaI/SalI.
6. method as claimed in claim 4 passes through at least 352 pairs of gRNA aligning primers of the essential gene of PAO1 bacterial strain
Gibson assembling, is cloned into respectively in pHERD20T-dCas9 plasmid, constructs the library CRISPRi.
7. method as claimed in claim 6, the library CRISPRi of the building be pHERD20T-dCas9-gRNA001 extremely
pHERD20T-dCas9-gRNA352。
8. the false list for the treatment of verdigris is being found in the pseudomonas aeruginosa CRISPRi system of the method building of claim 1 or 6 or library
The purposes of the antibiotic target spot of born of the same parents bacterium.
9. the pseudomonas aeruginosa CRISPRi system of the method building of claim 1 or 6 or library are for screening P. aeruginosa
The purposes of the indispensable gene function of bacterium.
10. the pseudomonas aeruginosa CRISPRi system of the method building of claim 1 or 6 or library are for regulating and controlling and inhibiting copper
The purposes of green pseudomonad indispensable gene expression.
Priority Applications (1)
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