Patent Application
20210032636
This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith:
The present application belongs to the field of genetic engineering, and relates to an improved promoter and a use thereof, in particular, to an improved promoter, a cloning vector with the improved promoter, a host cell with the cloning vector and uses thereof.
A polymerase chain reaction (PCR) technology is a major breakthrough in the fields of molecular biology and genetic engineering. After the PCR technology was developed, a technology for cloning PCR products into vectors (generally plasmids) has also been developed. Commonly-used and relatively simple cloning methods include TA cloning and blunt-end ligations. The PCR product amplified by Thermus aquaticus (Taq) enzymes contains a dAMP tail which can be ligated to a vector containing a T-terminus (T vector) under the action of T4 ligases, and this is the TA cloning. High-fidelity DNA polymerases generally contain 3′-5′ exonuclease activity, and the PCR products amplified by the high-fidelity DNA polymerases are blunt ends. These fragments are ligated to blunt-end vectors under the action of T4 ligases, which are the blunt-end ligations. These two methods have a common feature that the PCR products do not need to be treated in advance with special enzymes but are directly ligated to the vectors, which is simple and easily operated.
At present, commercially available T vectors and vectors that can be used for blunt-end cloning are generally based on the principle of blue-white screening. The blue-white screening is the most commonly used screening scheme to separate empty vectors from vectors with inserts. In this method, a reporter gene LacZα is used as a marker gene for the blue-white screening. However, vectors based on the principle of blue-white screening have the following problems during cloning: (1) due to the use of a strong promoter, the transcription and translation of foreign genes can be initiated in large quantities, which causes transcription or translation products of some foreign genes with complex structures to be toxic to hosts and cannot be cloned; (2) due to residual exonuclease activity of restriction enzymes when vectors are digested, repeated freezing and thawing of the digested vectors, long-term storage of digested linearized vectors and other factors, the prepared vectors lack 1-2 bases at digestion sites, leading to frameshift mutation of a lacZα gene, so that a clone without a foreign gene becomes white due to the frameshift mutation of the LacZα gene, resulting in a large number of false positive clones; (3) when a small foreign DNA fragment is cloned and a reading frame of the lacZα gene is not changed by inserting the foreign DNA, a false negative phenomenon that a plate is rich in blue spots will be caused; (4) when a foreign DNA fragment larger than 2 kb is cloned with a blunt-end vector, a few white spots and many blue spots are present, and the few white spots might grow together with the blue spots, so that white single clones are few, and it is difficult to select a sufficient number of positive clones. In addition, the blue-white screening further requires expensive and toxic chemical substances such as X-gal and IPTG.
Any DNA sequence that can be independently bound to a transcription factor and initiate transcription may be referred to as a promoter. A region recognizable by a σ factor in the promoter has very conserved sequence characteristics. Two sequences (referred to as −10 region and −35 region) about 10 nt and 35 nt upstream of a transcription starting site (+1) have a decisive effect on the recognition of the σ factor, so these two sequences are referred to as narrow promoters or core promoters. Other than the core promoters, sequences upstream of −35 region might also have an effect on transcription strength. These sequences are referred to as UP elements.
Therefore, the present application provides an improved promoter and a use thereof, so as to solve the problems that a prepared cloning vector fails in cloning, or a large number of false positive or negative clones are produced in the existing art.
To achieve the object, the present application adopts technical solutions described below.
In a first aspect, the present application provides an improved promoter. The improved promoter is obtained by mutating a nucleic acid sequence between −35 region and −10 region in a promoter region into recognition sites for an endonuclease.
In the present application, a change in the number of nucleic acids between −35 region and −10 region in a prokaryote will affect a level of gene transcription activity. The nucleic acid sequence between −35 region and −10 region in the promoter region is mutated to be recognized by the endonuclease. During cloning, a vector is prepared as a linearized vector, and then a foreign gene is ligated to the linearized vector, so that an expression-regulating gene of the promoter has decreased activity and a reduced expression amount, and then functions.
The recognition sites for the endonuclease refer to sites recognizable by any endonuclease, and the endonuclease is not limited and is selected mainly based on the convenience of experimental operations of those skilled in the art, as long as a successful mutation can be achieved by mutating one or several bases.
According to the present application, the improved promoter is obtained by mutating a nucleic acid sequence between −35 region and −10 region in a promoter region of a β-galactosidase into the recognition sites for the endonuclease.
In the present application, for a promoter of the β-galactosidase, the nucleic acid sequence between −35 region and −10 region in a strong promoter region is mutated into the recognition sites for the endonuclease that can be recognized, but it is cleaved into the linearized vector and inserted with a foreign fragment, so that a strong promoter of the β-galactosidase has significantly decreased activity due to the insertion of a foreign DNA fragment, an expression amount of a lacZα gene is significantly reduced, and a colony containing a recombinant plasmid is white, thereby overcoming the problem that a strong promoter in a vector based on blue-white screening initiates the transcription or translation of foreign genes and a transcription or translation product might be toxic to a host and cannot be cloned, avoiding the deficiency that frameshift mutation of the lacZα gene due to a lack of 1-2 bp of the vector at digestion sites results in false positive clones, and eliminating a false negative phenomenon that a plate is rich in blue spots due to a small fragment of foreign DNA and a reading frame of the lacZα gene which is unchanged by inserting the foreign DNA.
According to the present application, the nucleic acid sequence between −35 region and −10 region in the promoter region of the β-galactosidase is shown by SEQ ID NO.1-2, where nucleic acid sequences shown by SEQ ID NO.1-2 are as follows:
In the present application, an RNA polymerase II is generally bound at sites from −35 region to −10 region which are very important. An RNA polymerase can be in contact with a base in −35 and −10 sequences and a phosphate group in a primary DNA strand. A promoter farther from a common sequence has lower activity. The applicant has found that the foreign gene can be inserted by mutating a sequence from −35 region to −10 region, especially the sequence shown by SEQ ID NO.2, so as to significantly reduce the expression amount of the lacZα gene.
According to the present application, the endonuclease may be selected by those skilled in the art as required, and different recognition sites for the endonuclease may be selected according to different sequences to be mutated in the promoter region. In the present application, the endonuclease is selected from, but is not limited to, any one or a combination of at least two of EcoRV, AleI, BamHI, XhoI and PmlI.
According to the present application, a nucleic acid sequence between −35 region and −10 region of the improved promoter is shown by SEQ ID NO.3-14, where nucleic acid sequences shown by SEQ ID NO.3-14 are as follows:
In a second aspect, the present application provides a vector including the improved promoter described in the first aspect.
According to the present application, the vector further includes a gene of interest, where the gene of interest is operably ligated between the recognition sites for the endonuclease of the improved promoter.
In the present application, those skilled in the art may select the vector according to requirements. The selection of the vector will not affect a function of the promoter. The vector may be a cloning vector and/or an expression vector. The cloning vector is used for cloning a protein of interest, and the expression vector is used for expressing the protein of interest. The promoter may function on either the cloning vector or the expression vector. The vector is preferably the cloning vector, and the cloning vector may be, for example, a high-copy cloning vector pUC57, a low-copy cloning vector pCK or a single-copy cloning vector, each of which may carry the promoter of the present application, so as to carry out subsequent experiments without affecting the vector itself. The vector carrying the promoter of the present application is still a high-copy cloning vector, a low-copy cloning vector or a single-copy cloning vector.
In a third aspect, the present application provides a host cell, including the vector described in the second aspect.
According to the present application, the host cell is Escherichia coli, and a C-terminal ω-fragment of a β-galactosidase of the Escherichia coli is only encoded.
In the present application, a lacZα gene of the cloning vector encodes an N-terminal α-fragment of the β-galactosidase (lacZ), and the C-terminal ω-fragment of the β-galactosidase of the Escherichia coli is only encoded. Although none of encoded fragments of a host and a plasmid have galactosidase activity, when they exist at the same time, the α-fragment and the ω-fragment may form the β-galactosidase with enzymatic activity through α-complementation, and the β-galactosidase may cleave a colorless compound, X-gal(5-bromo-4-chloro-3-indole-β-D-galactoside), into galactose and a dark blue substance, 5-bromo-4-indigo which may make the whole colony appear blue. After foreign DNA is inserted into the promoter region of the β-galactosidase of the cloning vector of the present application, an expression amount of lacZα is significantly reduced, and a large amount of β-galactosidases with enzymatic activity cannot be effectively formed through the α-complementation, and eventually the colony appears white.
In a fourth aspect, the present application provides a method for preparing the vector described in the second aspect. The method includes steps described below.
(1) A primer is designed according to recognition sites for an endonuclease to be mutated into, and an original promoter and an expression-regulating gene of the original promoter are used as a template for PCR amplification, to obtain a product with an improved promoter.
(2) The product in step (1) is cyclized by a Gibson recombination method to obtain a vector with the promoter.
(3) The vector in step (2) is linearized.
(4) A gene of interest is ligated to the linearized vector in step (3) to obtain the vector.
According to the present application, a nucleic acid sequence of the primer in step (1) is shown by SEQ ID NO.15-38.
In the present application, in a plasmid constructed by performing PCR amplification on pUC57-lacZ or pCK-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.15-16, a nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.3.
In a plasmid constructed by performing PCR amplification on pUC57-lacZ or pCK-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.17-18, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.4.
In a plasmid constructed by performing PCR amplification on pUC57-lacZ or pCK-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.19-20, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.5.
In a plasmid constructed by performing PCR amplification on pUC57-lacZ or pCK-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.21-22, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.6.
In a plasmid constructed by performing PCR amplification on pUC57-lacZ or pCK-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.23-24, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.7.
In a plasmid constructed by performing PCR amplification on pUC57-lacZ or pCK-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.25-26, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.8.
In a plasmid constructed by performing PCR amplification on pUC57-lacZ or pCK-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.27-28, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.9.
In a plasmid constructed by performing PCR amplification on pCC1-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.29-30, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.10.
In a plasmid constructed by performing PCR amplification on pCC1-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.31-32, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.11.
In a plasmid constructed by performing PCR amplification on pCC1-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.33-34, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.12.
In a plasmid constructed by performing PCR amplification on pCC1-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.35-36, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.13.
In a plasmid constructed by performing PCR amplification on pCC1-lacZ with a primer pair of nucleic acid sequences shown by SEQ ID NO.37-38, the nucleic acid sequence shown by SEQ ID NO.2 is mutated into a nucleic acid sequence shown by SEQ ID NO.14.
According to the present application, the linearizing in step (3) is performed through endonuclease digestion and/or the PCR amplification.
According to the present application, before step (1), the method further includes performing codon optimization on the expression-regulating gene.
According to the present application, the expression-regulating gene is a lacZ gene whose nucleic acid sequence is shown by SEQ ID NO.39, where the nucleic acid sequence shown by SEQ ID NO.39 is as follows:
According to the present application, the lacZ gene is subjected to the codon optimization, and a nucleic acid sequence of the lacZ gene subjected to the codon optimization is shown by SEQ ID NO.40, where the nucleic acid sequence shown by SEQ ID NO.40 is as follows:
In a fifth aspect, the present application provides a method for preparing a protein of interest. The method includes a step described below.
The host cell described in the third aspect is cultivated under conditions suitable for an expression of the protein of interest to obtain the protein of interest; where a vector in the host cell is an expression vector, and the protein of interest is a protein encoded by a gene of interest.
In a sixth aspect, the present application provides a kit, including the improved promoter described in the first aspect, the vector described in the second aspect or the host cell described in the third aspect.
Compared with the existing art, the present application has beneficial effects described below.
(1) In the present application, the nucleic acid sequence between −35 region and −10 region in the promoter region is mutated to be recognized by the endonuclease. During cloning, the vector is prepared as the linearized vector, and then the foreign gene is ligated to the linearized vector, so that the promoter has decreased activity, the expression amount of the expression-regulating gene is reduced, and then the promoter functions.
(2) The present application has a significant effect on the promoter of the β-galactosidase. The nucleic acid sequence between −35 region and −10 region in the strong promoter region is mutated into the recognition sites for the endonuclease that can be recognized. When it is cleaved into the linearized vector and inserted with the foreign fragment, the strong promoter of the β-galactosidase has significantly decreased activity due to the insertion of the foreign DNA fragment, the expression amount of the lacZα gene is significantly reduced, and the colony containing the recombinant plasmid appear white.
(3) The present application can overcome the problem that the strong promoter in the vector based on the blue-white screening initiates the transcription or translation of foreign genes and the transcription or translation product might be toxic to the host and cannot be cloned, avoid the deficiency that the frameshift mutation of the lacZα gene due to the lack of 1-2 bp of the vector at digestion sites results in the false positive clones, and eliminate the false negative phenomenon that the plate is rich in blue spots due to the small fragment of foreign DNA and the reading frame of the lacZα gene which is unchanged by inserting the foreign DNA.
(4) A method for constructing the cloning vector of the present application is simple and easy to operate, has high efficiency, and can construct the cloning vector in a short time.
To further elaborate on the technical means adopted and the effects achieved in the present application, the technical solutions of the present application are further described below through specific embodiments, but the present application is not limited to the scope of the embodiments.
The present application adopts conventional techniques and methods in the fields of genetic engineering and molecular biology, and general reference literature provides definitions and methods known to those skilled in the art. However, those skilled in the art may adopt other conventional methods, experimental schemes and reagents in the art on the basis of the technical solutions described in the present application without being limited by specific embodiments of the present application.
Experiments without specific techniques or conditions noted in the examples are conducted according to techniques or conditions described in the literature in the art or a product specification. The reagents or instruments used without manufacturers are conventional products commercially available through proper channels.
LacZ gene: a gene widely used in gene expression regulation researches. An encoded β-galactosidase (β-gal) is a tetramer composed of 4 subunits and can catalyze a hydrolysis of lactose. The β-gal is relatively stable, appears blue when stained with X-Gal as a substrate, and is easy to detect and observe. Many advantages of the LacZ gene make it a commonly-used marker gene in genetic engineering experiments such as screening of transformed strains and β-galactosidase color test method, that is, blue-white screening.
LacZα gene: an N-terminal α-fragment for encoding the β-galactosidase (lacZ). The β-galactosidase with enzymatic activity may be formed through α-complementation and cleave a colorless compound, X-gal(5-bromo-4-chloro-3-indole-β-D-galactoside), into galactose and a dark blue substance, 5-bromo-4-indigo.
Endonuclease: an enzyme that can hydrolyze a phosphodiester bond inside a molecular chain to generate oligonucleotides among nucleic acid hydrolases.
PCR technology: a polymerase chain reaction, in which DNA is denatured in vitro at a high temperature of 95° C. to be single-stranded, a primer combines with a single strand at a low temperature (generally about 60° C.) based on a principle of complementary base pairing, the temperature is adjusted to an optimal reaction temperature of a DNA polymerase (about 72° C.) at which the DNA polymerase synthesizes a complementary strand along a direction from phosphate to five-carbon sugar (5′-3′). A PCR instrument based on polymerases is in fact a temperature control device and can control the temperature well between a denaturation temperature, a renaturation temperature and an extension temperature.
Materials:
Kanamycin-resistant pUC57 plasmid Genewiz Inc. Suzhou
pCK plasmid Genewiz Inc. Suzhou
Chloramphenicol-resistant pCC1TM plasmid EPICENTRE
Top10F′ competent cell Invitrogen
Restriction enzymes: EcoRV, AleI, BamHI, XhoI NEB
T4 DNA ligase NEB
lambdaDNA NEB
Gibson Assembly® Master Mix kit NEB
Primer synthesis Genewiz Inc. Suzhou
The codon optimization of the lacZα gene includes a step described below.
The codon optimization was conducted on the lacZα gene (SEQ ID NO.39) in a pUC57 plasmid using codon optimization software (developed by Genewiz Inc. Suzhou), where the optimized lacZα gene was synthesized by Genewiz Inc. Suzhou. A nucleotide sequence is shown by SEQ ID No.39, specifically:
A method for constructing the high-copy cloning vector includes specific steps described below.
(I) The lacZα gene of pUC57 (kanamycin resistance) was replaced with the optimized lacZα gene in Example 1, specifically including steps described below.
(1) The kanamycin-resistant pUC57 plasmid was used as a template and SEQ ID NO.41-42 were used as primers for PCR amplification. Specific sequences are as follows:
A PCR system is shown in Table 1.
One group uses water as a sample for negative control.
Reaction conditions are listed in Table 2.
(2) A PCR solution obtained in step (1) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product.
(3) The Gibson Assembly® Master Mix kit was used for ligating a PCR purified product obtained in step (2) and the codon-optimized lacZα gene obtained in Example 1. A ligation system is shown in Table 3.
A ligation condition was a ligation reaction of 1 h at 50° C.
(4) A ligation product obtained in step (3) was transformed into Top10F′ competent cells which were finally coated with a kanamycin-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. A blue single clone was picked and subjected to Sanger sequencing on the next day, and a plasmid with a correct sequence was reserved and named pUC57-lacZ.
(II) A sequence, 5′-CTTTATGCTTCCGGCTCG-3′, between −35 region and −10 region in a promoter region of the β-galactosidase of the pUC57-lacZ plasmid was mutated into a sequence recognizable by the endonuclease, which specifically includes steps described below.
(1) The pUC57-lacZ plasmid successfully constructed in step (I) was used as a template, and primers F1-EcoRV, R1-EcoRV, F2-EcoRV, R2-EcoRV, F3-EcoRV, R3-EcoRV, F4-EcoRV, R4-EcoRV, F5-EcoRV, R5-EcoRV, F6-AleI, R6-AleI, F7-BamHI-XhoI and R7-BamHI-XhoI (SEQ ID NO.15-SEQ ID NO.28) were used as primers for the PCR amplification. Specific sequences are listed in Table 4.
A specific PCR system is shown in Table 1, and reaction conditions are listed in Table 2.
(2) A PCR solution obtained in step (1) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product. The Gibson Assembly® Master Mix kit was used for a ligation reaction. A ligation system is shown in Table 5.
A ligation condition was a ligation reaction of 1 h at 50° C.
(3) Each ligation product obtained in step (2) was transformed into Top10F′ competent cells which were finally coated with a kanamycin-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. A blue single clone was picked and subjected to Sanger sequencing on the next day, and a plasmid with a correct sequence was reserved and separately named pUC57-lacZ-Mu-1 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-GATATCGCTTCCGGCTCG-3′, and the plasmid was constructed with primers F1-EcoRV+R1-EcoRV), pUC57-lacZ-Mu-2 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTGATATCTCCGGCTCG-3′, and the plasmid was constructed with primers F2-EcoRV+R2-EcoRV), pUC57-lacZ-Mu-3 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTATGATATCGGCTCG-3′, and the plasmid was constructed with primers F3-EcoRV+R3-EcoRV), pUC57-lacZ-Mu-4 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTATGCTGATATCTCG-3′, and the plasmid was constructed with primers F4-EcoRV+R4-EcoRV), pUC57-lacZ-Mu-5 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTATGCTTCCGATATC-3′, and the plasmid was constructed with primers F5-EcoRV+R5-EcoRV), pUC57-lacZ-Mu-6 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTCACCTTCGTGCTCG-3′, and the plasmid was constructed with primers F6-AleI+R6-AleI), and pUC57-lacZ-Mu-7 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTCGAGGATATCGGATCC-3′, and the plasmid was constructed with primers F7-BamHI-XhoI+R7-BamHI-XhoI).
(III) Vector Cloning Experiments
(1) The correct plasmids pUC57-lacZ-Mu-1, pUC57-lacZ-Mu-2, pUC57-lacZ-Mu-3, pUC57-lacZ-Mu-4, pUC57-lacZ-Mu-5 constructed in step (II) were digested with a restriction enzyme EcoRV, pUC57-lacZ-Mu-6 was digested with a restriction enzyme AleI, and pUC57-lacZ-Mu-7 was digested with restriction enzymes BamHI and XhoI. Digestion products were subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified.
(2) Reversely complementary primers of 24 bp and 48 bp were synthesized and annealed to form double-stranded DNA. Nucleotide sequences of the reversely complementary primers of 24 bp and 48 bp are shown by SEQ ID NO.43-SEQ ID NO.46, specifically:
(3) λ DNA was used as a template, and F-λDNA-200 bp+R-λDNA-200 bp were used as primers for the PCR amplification. Nucleotide sequences of the primers F-λDNA-200 bp and R-λDNA-200 bp are shown by SEQ ID NO.47-SEQ ID NO.48, specifically:
A PCR system is shown in Table 1, and a PCR amplification program is shown in Table 6.
(4) A PCR solution obtained in step (3) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product. The purified PCR amplification product was digested with BamHI and XhoI. A digestion system is shown in Table 7.
A digestion condition was digestion of 1 h at 37° C., and the digestion product was recovered and purified using an Axygen purification kit.
(5) Fragments of 24 bp and 48 bp formed after annealing in step (2) were ligated to the digested and purified vectors in step (1), pUC57-lacZ-Mu-1, pUC57-lacZ-Mu-2, pUC57-lacZ-Mu-3, pUC57-lacZ-Mu-4, pUC57-lacZ-Mu-5 and pUC57-lacZ-Mu-6 separately, and the purified digestion product in step (4) was ligated to the digested and purified vector in step (1), pUC57-lacZ-Mu-7. A ligation system is shown in Table 8.
A ligation condition was a ligation reaction of 1 h at 22° C.
(6) Each ligation product obtained in step (5) was transformed into Top10F′ competent cells which were finally coated with the kanamycin-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. 24 white single clones were picked on the plate of the cloned DNA fragment of about 200 bp (pUC57-lacZ-Mu-7 vector) for colony PCR identification on the next day. The PCR system is shown in Table 9.
A PCR amplification program is shown in Table 10.
A PCR identification result is shown in
The result in
A method for constructing the pUC57-lacZ-Mu-2 plasmid includes steps described below.
(1) The plasmid pUC57-lacZ-Mu-2 constructed in Example 2 was used as a template, and F1-del+R1-del, F2-del+R2-del and F3-del+R3-del were used as primers for PCR amplification. Nucleotide sequences of the primers F1-del, R1-del, F2-del, R2-del, F3-del and R3-del are shown by SEQ ID NO.49-SEQ ID NO.54, specifically:
A PCR system is shown in Table 1 in Example 2, and a PCR amplification program is shown in Table 2 in Example 2.
(2) A PCR solution obtained in step (1) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product. The Gibson Assembly® Master Mix (NEB) kit was used for a ligation reaction. A ligation system is shown in Table 5 in Example 2.
A ligation condition was a ligation reaction of 1 h at 50° C.
(3) Each ligation product obtained in step (2) was transformed into Top10F′ competent cells which were finally coated with a kanamycin-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. Blue single clones were picked and subjected to Sanger sequencing on the next day, and plasmids with correct sequences were reserved to obtain the three mutant plasmids of the pUC57-lacZ-Mu-2, which are named pUC57-lacZ-Mu-2A, pUC57-lacZ-Mu-2B and pUC57-lacZ-Mu-2C, separately.
An EcoRV site of pUC57-lacZ-Mu-2A was mutated into GATTC, that is, the sequence was mutated from 5′-CTTGATATCTCCGGCTCG-3′ (SEQ ID NO.59) to 5′-CTTGATTCTCCGGCTCG-3′ (SEQ ID NO.60). An EcoRV site of pUC57-lacZ-Mu-2B was mutated into GAATC, that is, the sequence was mutated from 5′-CTTGATATCTCCGGCTCG-3′ to 5′-CTTGAATCTCCGGCTCG-3′(SEQ ID NO.61). An EcoRV site of pUC57-lacZ-Mu-2C was mutated into GATC, that is, the sequence was mutated from 5′-CTTGATATCTCCGGCTCG-3′ to 5′-CTTGATCTCCGGCTCG-3′ (SEQ ID NO.62).
(4) Correct plasmids pUC57-lacZ-Mu-2A, pUC57-lacZ-Mu-2B and pUC57-lacZ-Mu-2C in step (3) each were transformed into Top10F′ competent cells which were finally coated with the kanamycin-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. It was found on the next day that colonies on three plates were all blue. 5 single clones were selected from each plate and subjected to Sanger sequencing. Sequencing results show that all clones have correct sequences.
Experimental results show that β-galactosidase promoters of the three mutant plasmids of pUC57-lacZ-Mu-2 (pUC57-lacZ-Mu-2A, pUC57-lacZ-Mu-2B and pUC57-lacZ-Mu-2C) still have activity and can express lacZα and make colonies appear blue when induced by IPTG. Meanwhile, the experimental results show that the linearized pUC57-lacZ-Mu-2 vector after EcoRV digestion can still express lacZα and make colonies appear blue after the self-ligation of the linearized vector that lacks 1 base at one end of the site (pUC57-lacZ-Mu-2A and pUC57-lacZ-Mu-2B plasmids) or two ends of the site (pUC57-lacZ-Mu-2C plasmid), that is, the vector of the present application, after digested by the endonuclease, will not generate false positive clones due to the self-ligation for the lack of 1 base at one end or two ends of the site.
A method for constructing the low-copy cloning vector includes specific steps described below.
(I) The lacZα gene of pCK plasmid was replaced with the optimized lacZα gene in Example 1, specifically including steps described below.
(1) The pCK plasmid was used as a template and SEQ ID NO.55-56 were used as primers for PCR amplification. Specific sequences are as follows:
A PCR system is shown in Table 1 in Example 2, and reaction conditions are shown in Table 2 in Example 2.
(2) A PCR solution obtained in step (1) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product.
(3) The Gibson Assembly® Master Mix kit was used for ligating a PCR purified product obtained in step (2) and the codon-optimized lacZα gene obtained in Example 1. A ligation system is shown in Table 3 in Example 2.
A ligation condition was a ligation reaction of 1 h at 50° C.
(4) A ligation product obtained in step (3) was transformed into Top10F′ competent cells which were finally coated with a kanamycin-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. A blue single clone was picked and subjected to Sanger sequencing on the next day, and a plasmid with a correct sequence was reserved and named pCK-lacZ.
(II) A sequence, 5′-CTTTATGCTTCCGGCTCG-3′, between −35 region and −10 region in a promoter region of the β-galactosidase of the pCK-lacZ plasmid was mutated into a sequence recognizable by the endonuclease, which specifically includes steps described below.
(1) The pCK-lacZ plasmid successfully constructed in step (I) was used as a template, and primers F1-EcoRV, R1-EcoRV, F2-EcoRV, R2-EcoRV, F3-EcoRV, R3-EcoRV, F4-EcoRV, R4-EcoRV, F5-EcoRV, R5-EcoRV, F6-AleI, R6-AleI, F7-BamHI-XhoI and R7-BamHI-XhoI (SEQ ID NO.15-SEQ ID NO.28) were used as primers for the PCR amplification. Specific sequences are listed in Table 4 in Example 2. A specific PCR system is shown in Table 1 in Example 2, and reaction conditions are shown in Table 2 in Example 2.
(2) A PCR solution obtained in step (1) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product. The Gibson Assembly® Master Mix kit was used for a ligation reaction. A ligation system is shown in Table 5 in Example 2.
A ligation condition was a ligation reaction of 1 h at 50° C.
(3) Each ligation product obtained in step (2) was transformed into Top10F′ competent cells which were finally coated with the kanamycin-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. A blue single clone was picked and subjected to Sanger sequencing on the next day, and a plasmid with a correct sequence was reserved and separately named pCK-lacZ-Mu-1 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-GATATCGCTTCCGGCTCG-3′, and the plasmid was constructed with primers F1-EcoRV+R1-EcoRV), pCK-lacZ-Mu-2 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTGATATCTCCGGCTCG-3′, and the plasmid was constructed with primers F2-EcoRV+R2-EcoRV), pCK-lacZ-Mu-3 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTATGATATCGGCTCG-3′, and the plasmid was constructed with primers F3-EcoRV+R3-EcoRV), pCK-lacZ-Mu-4 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTATGCTGATATCTCG-3′, and the plasmid was constructed with primers F4-EcoRV+R4-EcoRV), pCK-lacZ-Mu-5 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTATGCTTCCGATATC-3′, and the plasmid was constructed with primers F5-EcoRV+R5-EcoRV), pCK-lacZ-Mu-6 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTCACCTTCGTGCTCG-3′, and the plasmid was constructed with primers F6-AleI+R6-AleI), and pCK-lacZ-Mu-7 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTCGAGGATATCGGATCC-3′, and the plasmid was constructed with primers F7-BamHI-XhoI+R7-BamHI-XhoI).
(III) Vector Cloning Experiments
(1) The correct plasmids pCK-lacZ-Mu-1, pCK-lacZ-Mu-2, pCK-lacZ-Mu-3, pCK-lacZ-Mu-4 and pCK-lacZ-Mu-5 constructed in step (II) were digested with a restriction enzyme EcoRV, pCK-lacZ-Mu-6 was digested with a restriction enzyme AleI, and pCK-lacZ-Mu-7 was digested with restriction enzymes BamHI and XhoI. Digestion products were subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified.
(2) Reversely complementary primers of 24 bp and 48 bp were synthesized and annealed to form double-stranded DNA. Nucleotide sequences of the reversely complementary primers of 24 bp and 48 bp are shown by SEQ ID NO.43-SEQ ID NO.46 in Example 2.
(3) λ DNA was used as a template, and F-λDNA-200 bp+R-λDNA-200 bp were used as primers for the PCR amplification. Nucleotide sequences of the primers F-λDNA-200 bp and R-λDNA-200 bp are shown by SEQ ID NO.47-SEQ ID NO.48 in Example 2.
A PCR system is shown in Table 1 in Example 2, and a PCR amplification program is shown in Table 6 in Example 2.
(4) A PCR solution obtained in step (3) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product. The purified PCR amplification product was digested with BamHI and XhoI. A digestion system is shown in Table 7 in Example 2.
A digestion condition was digestion of 1 h at 37° C., and the digestion product was recovered and purified using an Axygen purification kit.
(5) Fragments of 24 bp and 48 bp formed after annealing in step (2) were ligated to the digested and purified vectors in step (1), pCK-lacZ-Mu-1, pCK-lacZ-Mu-2, pCK-lacZ-Mu-3, pCK-lacZ-Mu-4, pCK-lacZ-Mu-5 and pCK-lacZ-Mu-6, separately, and the purified digestion product in step (4) was ligated to the digested and purified vector in step (1), pCK-lacZ-Mu-7. A ligation system is shown in Table 8 in Example 2.
A ligation condition was a ligation reaction of 1 h at 22° C.
(6) Each ligation product obtained in step (5) was transformed into Top10F′ competent cells which were finally coated with the kanamycin-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. 12 white single clones were picked on the plate of the cloned DNA fragment of about 200 bp (pCK-lacZ-Mu-7 vector) for colony PCR identification on the next day. The PCR system is shown in Table 9.
A PCR amplification program is shown in Table 10.
A result in
A method for constructing the single-copy cloning vector includes specific steps described below.
(I) The lacZα gene of pCC1 plasmid was replaced with the optimized lacZα gene in Example 1, specifically including steps described below.
(1) The pCC1 plasmid was used as a template and SEQ ID NO.57-58 were used as primers for PCR amplification. Specific sequences are as follows:
A PCR system is shown in Table 1 in Example 2, and reaction conditions are shown in Table 11.
(2) A PCR solution obtained in step (1) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product.
(3) The Gibson Assembly® Master Mix kit was used for ligating a PCR purified product obtained in step (3) and the codon-optimized lacZα gene obtained in Example 1. A ligation system is shown in Table 12 in Example 2.
A ligation condition was a ligation reaction of 1 h at 50° C.
(4) A ligation product obtained in step (3) was transformed into Top10F′ competent cells which were finally coated with a chloramphenicol-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. A blue single clone was picked and subjected to Sanger sequencing on the next day, and a plasmid with a correct sequence was reserved and named pCC1-lacZ.
(II) A sequence, 5′-CTTTATGCTTCCGGCTCG-3′, between −35 region and −10 region in a promoter region of the β-galactosidase of the pCC1-lacZ plasmid was mutated into a sequence recognizable by the endonuclease, which specifically includes steps described below.
(1) The pCC1-lacZ plasmid successfully constructed in step (I) was used as a template, and primers F1-PmlI+R1-PmlI, F2-PmlI+R2-PmlI, F3-PmlI+R3-PmlI, F4-PmlI+R4-PmlI, F5-PmlI+R5-PmlI and F7-BamHI-XhoI+R7-BamHI-XhoI (SEQ ID NO.29-SEQ ID NO.38, SEQ ID NO.27-SEQ ID NO.28) were used as primers for the PCR amplification. Specific sequences are listed in Table 13.
A specific PCR system is shown in Table 1 in Example 2, and reaction conditions are shown in Table 11.
(2) A PCR solution obtained in step (1) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product. The Gibson Assembly® Master Mix kit was used for a ligation reaction. A ligation system is shown in Table 14 in Example 2.
A ligation condition was a ligation reaction of 1 h at 50° C.
(3) Each ligation product obtained in step (2) was transformed into Top10F′ competent cells which were finally coated with the chloramphenicol-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. A blue single clone was picked and subjected to Sanger sequencing on the next day, and a plasmid with a correct sequence was reserved and separately named pCC1-lacZ-Mu-1 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CACGTGGCTTCCGGCTCG-3′, and the plasmid was constructed with primers F1-PmlI+R1-PmlI), pCC1-lacZ-Mu-2 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTCACGTGTCCGGCTCG-3′, and the plasmid was constructed with primers F2-PmlI+R2-PmlI), pCC1-lacZ-Mu-3 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTATCACGTGGGCTCG-3′, and the plasmid was constructed with primers F3-PmlI+R3-PmlI), pCC1-lacZ-Mu-4 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTATGCTCACGTGTCG-3′, and the plasmid was constructed with primers F4-PmlI+R4-PmlI), pCC1-lacZ-Mu-5 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTTTATGCTTCCCACGTG-3′, and the plasmid was constructed with primers F5-PmlI+R5-PmlI), and pCC1-lacZ-Mu-6 (5′-CTTTATGCTTCCGGCTCG-3′ was mutated into 5′-CTCGAGGATATCGGATCC-3′, and the plasmid was constructed with primers F7-BamHI-XhoI+R7-BamHI-XhoI).
(III) Vector Cloning Experiments
(1) The correct plasmids pCC1-lacZ-Mu-1, CC1-lacZ-Mu-2, pCC1-lacZ-Mu-3, pCC1-lacZ-Mu-4 and pCC1-lacZ-Mu-5 constructed in step (II) were digested with a restriction enzyme PmlI, and pCC1-lacZ-Mu-6 was digested with restriction enzymes BamHI and XhoI. Digestion products were subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified.
(2) Reversely complementary primers of 24 bp and 48 bp were synthesized and annealed to form double-stranded DNA. Nucleotide sequences of the reversely complementary primers of 24 bp and 48 bp are shown by SEQ ID NO.43-SEQ ID NO.46 in Example 2.
(3) λ DNA was used as a template, and F-λDNA-200 bp+R-λDNA-200 bp were used as primers for the PCR amplification. Nucleotide sequences of the primers F-λDNA-200 bp and R-λDNA-200 bp are shown by SEQ ID NO.47-SEQ ID NO.48 in Example 2.
A PCR system is shown in Table 1, and a PCR amplification program is shown in Table 6.
(4) A PCR solution obtained in step (3) was subjected to 1% agarose gel electrophoresis, and gel was cut, recovered and purified to obtain a PCR amplification product. The purified PCR amplification product was digested with BamHI and XhoI. A digestion system is shown in Table 7 in Example 2.
A digestion condition was digestion of 1 h at 37° C., and the digestion product was recovered and purified using an Axygen purification kit.
(5) Fragments of 24 bp and 48 bp formed after annealing in step (2) were ligated to the digested and purified vectors in step (1), pCC1-lacZ-Mu-1, pCC1-lacZ-Mu-2, pCC1-lacZ-Mu-3, pCC1-lacZ-Mu-4 and pCC1-lacZ-Mu-5, separately, and the purified digestion product in step (4) was ligated to the digested and purified vector in step (1), pCC1-lacZ-Mu-6. A ligation system is shown in Table 7 in Example 2.
A ligation condition was a ligation reaction of 1 h at 22° C.
(6) Each ligation product obtained in step (5) was transformed into Top10F′ competent cells which were finally coated with the chloramphenicol-resistant LB plate containing IPTG and X-gal and cultivated overnight at 37° C. 24 white single clones were picked on the plate of the cloned DNA fragment of about 200 bp (pCC1-lacZ-Mu-6 vector) for colony PCR identification on the next day. The PCR system is shown in Table 9.
A PCR amplification program is shown in Table 10. A result is shown in
An electrophoresis result in
To conclude, in the present application, a sequence between −35 region and −10 region in a strong promoter region of the β-galactosidase is mutated into sites that can be recognized and digested by the endonuclease, and during cloning, a vector is digested with an appropriate endonuclease or a linearized vector is prepared by a PCR method, and then the linearized vector is ligated to foreign genes, so that a strong promoter of the β-galactosidase has significantly decreased activity due to the insertion of a foreign DNA fragment, an expression amount of the lacZα gene is significantly reduced, and a colony containing a recombinant plasmid is white. By use of the above-mentioned method, the present application overcomes the common problem that a strong promoter in a vector based on blue-white screening initiates the transcription or translation of foreign genes and a transcription or translation product might be toxic to a host and cannot be cloned, can avoid the deficiency that frameshift mutation of the lacZα gene due to a lack of 1-2 bp of the vector at digestion sites results in false positive clones, and can eliminate a false negative phenomenon that a plate is rich in blue spots due to a small fragment of foreign DNA and a reading frame of the lacZα gene which is unchanged by inserting the foreign DNA.
The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosed scope of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201711490227.9 | Dec 2017 | CN | national |
This application is a continuation-in-part of International Application No. PCT/CN2018/122309, which designated the United States and was filed on Dec. 20, 2018, published in Chinese, which claims priority under 35 U.S.C. § 119 or 365 to CN Application No. 201711490227.9, filed Dec. 29, 2017. The entire teachings of the above applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2018/122309 | Dec 2018 | US |
| Child | 16914266 | US |
