CN108841901B - Kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 and application thereof - Google Patents
Kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 and application thereof Download PDFInfo
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Abstract
本发明公开了一种依赖T5核酸外切酶和PEG8000完成DNA组装的试剂盒，是由T5核酸外切酶和含PEG8000的缓冲体系组成；以总体系为20uL计，T5核酸外切酶的用量是0.02U‑0.16U/总反应体系，PEG8000的缓冲体系组分为110±5mM、pH＝7.5的Tris‑HCl，10±1mM MgCl2，10±1mM DTT和质量比5±1％的PEG8000。本发明还公开了所述试剂盒在DNA重组无缝克隆中的应用，实验证实本发明的试剂盒和相关的DNA组装方法与已经商业化的组装试剂具有等同的精确、高效和便捷的特点，但却比目前商业化试剂盒的售价低100倍，较现有方法更为经济和实用。
The invention discloses a kit for completing DNA assembly relying on T5 exonuclease and PEG8000. It is 0.02U-0.16U/total reaction system, and the buffer system components of PEG8000 are 110±5mM, pH=7.5 Tris-HCl, 10±1mM MgCl 2 , 10±1mM DTT and PEG8000 with a mass ratio of 5±1%. The invention also discloses the application of the kit in the seamless cloning of DNA recombination. Experiments have confirmed that the kit and the related DNA assembly method of the invention have the same characteristics of accuracy, efficiency and convenience as the commercialized assembly reagents. However, it is 100 times cheaper than the current commercial kit, and is more economical and practical than the existing method.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a kit for DNA assembly and application thereof, and particularly relates to a cheap kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 and application thereof.
Background
DNA cloning was a technique invented 40 years ago, and fusion of DNA molecules was accomplished by enzymatic cleavage and ligation, and has milestone significance for the development of molecular biology, which is still the most widely known method at present. However, with the advent and development of metabolic engineering and synthetic biology, the construction and modification of genetic modules has become more demanding on cloning methods. The inefficiency, sequence dependence and non-modeling of the digestion and ligation methods have made it unsuitable for current operations, and in recent years, new techniques for multiple DNA assembly have emerged, overcoming the limitations of traditional digestion and ligation methods.
These methods can be classified into restriction enzyme-dependent cloning, in vitro recombination-dependent cloning, in vivo recombination-dependent cloning, and short-fragment splicing cloning, according to the mechanism by which they operate. The in vitro recombination cloning method is mainly divided into assembly completed by means of Polymerase Chain Reaction (PCR) and assembly completed by means of short homologous arm recombination. Wherein, the assembly completed by the method relying on the short homologous recombination has the characteristics of independent sequence and independent connection, and the selection is more free. Designing a short homologous region (with the length of 15bp-50bp being different) with completely same sequence at two molecular terminals in vitro, cutting single-chain terminals by means of different enzymes, freely annealing complementary single-chain terminals, completing in vitro repair or directly transforming cells with the assistance of other enzyme systems, completing DNA repair with the assistance of in vivo enzyme systems, and further completing the purpose of DNA cloning. Currently common assembly methods can be divided into two broad categories depending on the mechanism: one is by means of the 3 '-5' exonuclease activity of high fidelity DNA polymerase, which 3 '-5' exonuclease activity can cut DNA into 5 '-single-stranded ends without adding dNTPs, and this spontaneous annealing between DNA molecules with 5' single-stranded ends forms circular DNA with single-stranded nicks, after which this molecule is repaired intracellularly to obtain complete plasmid. The other is a famous Gibson DNA assembling method, firstly, a DNA molecule is cut into a 3 'single-stranded end by means of T5 exonuclease, then the 3' single-stranded end is spontaneously annealed to form intermolecular collocation, then the repair of the deletion DNA is completed under the action of Phusion DNA polymerase, then the connection is completed under the action of Taq DNA ligase, further the DNA molecule repair is completed extracellularly to form a complete DNA molecule, then, people make an improvement on the basis of the Gibson assembling technology, and the Taq DNA ligase and the cofactor NADH thereof are not necessary when cloning is carried out, so that the method is named as a Hot-Phusion technology. In addition, the applicant of the present patent also noted that the near-shore protein science and technology company applied for a patent (application publication No.: CN 107760706A); the patent mentions that assembly can be performed using only T5 exonuclease, giving an effective concentration of T5 exonuclease between 0.01U and 20U. The buffer system used is 10-100 mM KAc, 20-100 mM Tris-Ac, 1-20 mM Mg (Ac)2 and 1-5 mM DTT, the reaction temperature is 25 ℃ or 37 ℃, the reaction time is 10-15min, but the overall titer is lower as a result of the implementation.
The current various assembling methods are infinite, have advantages and disadvantages and are most suitable for the field, but the current in-vitro assembling method is expensive in whole price or low in efficiency, and is not suitable for large-scale popularization and application. Accompanied by synthesisThe development of the field of biology puts higher demands on cloning technology, and the accurate, efficient, simple and economical cloning method is more beneficial to the development of synthetic biology. Through the search, a method for completing DNA assembly by constructing a small amount of T5 exonuclease with the help of PEG8000 and optimizing the conditions, TEDA (named TEDA assembly,T5 Exonuclease Dependent Assembly), and related kits that rely on T5 exonuclease and PEG8000 to accomplish DNA assembly have not been reported.
Disclosure of Invention
In view of the defects of the existing DNA recombination seamless cloning method, the invention aims to provide a kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 and application thereof.
The kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 is characterized in that: the kit consists of T5 exonuclease and a buffer system containing PEG 8000; the dosage of T5 exonuclease is 0.02U-0.16U/total reaction system, and the buffer system component of PEG8000 is 110 + -5 mM Tris-HCl with pH 7.5, 10 + -1 mM MgCl 210. + -. 1mM DTT and 5. + -. 1% by mass of PEG 8000.
In the above kit for completing DNA assembly by relying on T5 exonuclease and PEG 8000: the kit is calculated by 20uL of total system, the dosage of the T5 exonuclease is preferably 0.04U-0.08U/total reaction system, and the buffer system component of PEG8000 is 105-110 mM Tris-HCl with pH 7.5, 10mM MgCl210mM DTT and 5% by mass of PEG 8000.
In the above kit for completing DNA assembly by relying on T5 exonuclease and PEG 8000: the kit is calculated by 20uL of total system, the most preferable dosage of the T5 exonuclease is 0.04U/total reaction system, the buffer system component of PEG8000 is 105mM Tris-HCl with pH 7.5, 10mM MgCl210mM DTT and 5% by mass of PEG 8000.
The components in the kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 allow pre-configuration of concentration of 4/3, 2 or 5, reserve for later addition of DNA, and the concentration condition does not influence the effect of assembly reaction. T5 exonuclease and reaction buffer are mixed in the kit in advance or added later.
The kit is tested to be stored at-20 ℃ and-80 ℃ without influencing the reaction effect; and the kit can be stored at low temperature for more than half a year without influencing the reaction effect.
The kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 is applied to DNA recombination seamless cloning.
Wherein, the seamless cloning method comprises the following steps:
(1) linearizing the plasmid vector by a PCR method or a restriction enzyme cutting method;
(2) introducing homologous arm sequences homologous to two ends of the plasmid vector into two ends of the target gene fragment through PCR primer design, and obtaining the target gene fragment through PCR amplification;
(3) the kit which completes DNA assembly by relying on T5 exonuclease and PEG8000 is used for uniformly mixing the linearized plasmid vector and the linearized target gene obtained by amplification for assembly reaction;
(4) transforming the assembly system in the step (3) into competent cells of escherichia coli for verification;
the method is characterized in that:
the length of the homologous arm sequence in the step (2) is more than 9 bp;
the assembly reaction in the step (3) is carried out for 10min to 60min at the temperature of 15 ℃ to 60 ℃; the dosage of the linear plasmid with 3 Kb-6 Kb is 50ng-400ng, and the molar ratio of the linear vector to the insert is 8:1-1: 8;
preparing competent cells of the transformed escherichia coli in the step (4) by adopting a KCM method and an Inoue method; competent cells were strain DH5 α, JM109, Top10, XL-1 Blue MRF ', GB05, S17-1, CC118, SM10 or XL-1 Blue MRF'.
In the above application, a preferred embodiment is:
the length of the homologous arm sequence in the step (2) is 9bp-50 bp;
the assembly reaction in the step (3) is carried out for 40-60 min at the temperature of 25-37 ℃; the linear plasmid dosage of 3Kb is 25ng-200ng (if the linear plasmid is longer, the molecular weight is predicted to be larger, the content of DNA can be improved in an equal proportion, if the linear plasmid is 6Kb, the dosage is controlled to be 50ng-400ng), and the molar ratio of the linear plasmid to the insert fragment is 1:1-1: 4;
preparing competent cells of the transformed escherichia coli in the step (4) by adopting an Inoue method; the competent cells were E.coli strain DH5 alpha or XL-1 Blue MRF'.
In the above application, a further preferred embodiment is:
the length of the homologous arm sequence in the step (2) is 9bp-30 bp;
the assembly reaction in the step (3) is carried out for 40 plus or minus 2min at the temperature of 30 plus or minus 2 ℃; the dosage of the linear plasmid with 3Kb is 100ng-200ng, and the molar ratio of the linear vector to the insert is 1:1-1: 2;
preparing competent cells of the transformed escherichia coli in the step (4) by adopting an Inoue method; the competent cells were E.coli strain DH5 alpha or XL-1 Blue MRF'.
In the above applications, the most preferred embodiments are:
the length of the homologous arm sequence in the step (2) is 15bp-30 bp;
the assembly reaction in the step (3) is carried out for 40min at the temperature of 30 ℃; the dosage of the linear plasmid with 3Kb is 200ng, and the molar ratio of the linear vector to the insert is 1: 1;
preparing competent cells of the transformed escherichia coli in the step (4) by adopting an Inoue method; the competent cells were E.coli strain XL-1 Blue MRF'.
And (3) experimental test: coli strains DH5 alpha and XL-1 Blue MRF' were the best, and competent cells prepared by Inoue method were more favorable for TEDA assembly because of high transformation titer.
The invention discloses a kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 and a DNA assembly method TEDA (A/D)T5 Exonuclease Dependent Assembly). Through primer design and PCR amplification, two ends of the insert fragment are respectively provided with the same tail ends as the two ends of the linear vector; in a system containing T5 exonuclease, reacting at 30 ℃ for 40min to cut the fragments and plasmids and expose the linear plasmid vectorAnd the complementary 3' -protruding linear ends of the insert spontaneously anneal to form a circular DNA molecule with staggered single-stranded nicks; PEG8000 molecules are added into the system, so that the annealing matching effect is further enhanced; then the molecule with the special structure is transferred into cells to complete the repair by virtue of a repair system in the cells; the specific principle is shown in fig. 1.
The kit and the related DNA assembly method have the characteristics of accuracy, high efficiency and convenience which are equivalent to those of the commercialized assembly reagent, but the cost of the kit is 100 times lower than the sale price of the current commercialized kit, and the cost of the kit is the lowest among the reported assembly methods and is 10 times lower than the lowest cost of the method reported by the current published documents. The method of the invention firstly provides that T5 exonuclease and PEG8000 can be directly used for conversion to complete assembly, and the method is more efficient, convenient and economical than the existing method. Although the technical scheme of the invention mainly adopts T5 exonuclease, the applicant finds that the addition of PEG8000 can obviously improve the assembly efficiency, and the whole assembly efficiency is improved by more than 10 times. In addition, the invention optimizes the enzyme concentration, Buffer components, reaction time, reaction temperature, DNA dosage, homology arm length, selection of escherichia coli cells, preparation method of competent cells, system concentration ratio, storage temperature, storage time and the like, and produces unexpected effects.
The invention has the beneficial effects that:
1) the reaction is cut by means of T5 exonuclease, PEG8000 has protection effect on the annealing of DNA molecules, and the assembly effect is improved;
2) the TEDA assembly product can be directly transformed into escherichia coli cells without subsequent operation;
3) the reaction does not need the assistance of a complex enzyme system, and has the advantages of simple system, low cost, convenient operation, easy large-scale high-flux operation, short reaction time, low mutation rate, high reaction efficiency and high fidelity. The assembly of the assembled DNA molecules can be completed rapidly;
4) the TEDA assembly positive rate is high, and more than 95% positive clones can be obtained;
5) TEDA assembly can complete the assembly of multiple DNA fragments simultaneously;
6) TEDA assembly is also applied to site-directed mutagenesis of target DNA, and site-directed mutagenesis of multiple sites can be simultaneously accomplished.
Drawings
FIG. 1 shows TEDA (CT5 Exonuclease Dependent Aschematic) principle schematic of the assembly technique.
FIG. 2 shows the assembly of 1-3 fragments with TEDA and a linear plasmid vector.
Wherein: the phbCAB gene cluster for synthesizing PHB is a multiple cistron structure and sequentially comprises 3 genes: phbC, phbA and phbB, placed under the promoter of 5-tac tandem to obtain P5tac-phbCAB fragment. Dividing P5tac-phbCAB into 3 fragments by PCR with the internal sites of the ORF of phbC and phbA genes as boundaries, namely an A fragment (containing a P5tac promoter region and 5 'end phbC, 1409bp), a B fragment (containing 3' end phbC and 5 'end phbA, 1363bp) and a C fragment (containing 3' end phbA and full-length phbB, 1685 bp); or into two segments, AB and C, or A and BC; or cloned as a complete fragment, named ABC. The pBluescript SK-plasmid is cut by SmaI enzyme to be used as a linearized plasmid vector with the length of 2958bp and is named as V. Linear plasmid vectors were used for single insert ABC, double insert AB + C or A + BC, 3 segment A + B + C, respectively. When the fragments are amplified by PCR through primer design, 20bp homology arms are introduced between the fragments or between the fragments and plasmids so as to facilitate the assembly. V concentration was then controlled at 100ng, insert: the molar ratio of the linearized plasmid vector is 1:1, a TEDA assembly reaction system is configured, the operation is carried out for 40min at the temperature of 30 ℃, then 5uL is selected for conversion, and the effect of the assembly reaction is tested.
Figure 3 shows the effect of PEG8000 on assembly in TEDA assembly.
Wherein: SmaI cut pSK linear plasmid and eGFP gene ORF are used as insertion fragments, 20bp homologous arms are designed at the tail ends of the pSK linear plasmid and the eGFP gene for assembly, and the assembly effect is tested.
Figure 4 tests the effect of using different T5 exonuclease concentrations on assembly.
Wherein: the reaction system used for the test was 100mM Tris-HCl (pH 7.5),10mM MgCl210mM DTT and 5% PEG8000, at 30 deg.C for 40 min. The linear vector used was SmaI cut pSK, the insert was the ORF of the eGFP gene, the amount of linear plasmid was controlled at 100ng and the molar ratio of insert to linear plasmid vector was 2: 1.
FIG. 5 shows the TEDA assembly method for mutation of DNA sites.
The P5tac-phbCAB fragment and pBBR1MCS-2 plasmid were first constructed into pBBR1MCS2-P5 tac-phbCAB. 3 Terminators (TAA) were inserted inside the phbC, phbB and phbA genes, creating pBBR1MCS2-P5tac-phbCAB-1TAA, pBBR1MCS2-P5tac-phbCAB-2TAA and pBBR1MCS2-P5tac-phbCAB-3TAA plasmids. Primers are designed at the TAA insertion sites respectively, and TAA sequences are removed from the primers to create corresponding homologous arm sequences. The TEDA assembly reaction was then run to test the efficiency of TEDA assembly to remove 1-3 stop codons at once.
FIG. 6 shows the TEDA assembly system configured at different concentrations and stored at-20 ℃ and-80 ℃ for different periods of time, and then tested for their effect on the assembly reaction.
Wherein: the plasmid model of lacZ was used pBBR1MCS 5. The ORF region of the lacZ gene of E.coli was first cloned, placed under the Plac promoter of pBBR1MCS5, and the complete lacZ α region on the pBBR1MCS-5 plasmid was replaced to construct pBBR1MCS5:, lacZ plasmid. The middle region of the ORF of the lacZ gene (Middke-lacZ,1937bp length, 17bp to 1953bp region of ORF) and pBBR1MCS5:: the remainder of the lacZ plasmid (pBBR1MCS5:: Part-lacZ) were each primed for PCR extension and 20bp homologous regions were designed in the corresponding primer regions. Two-fragment assembly was tested using the linearized plasmid pBBR1MCS5 Part-lacZ and the insert middlke-lacZ for TEDA assembly.
Detailed Description
In order to more clearly illustrate the technical solutions and embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the description of specific technical methods: it is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In some embodiments, linear plasmid (3Kb) concentrations between 50ng and 400ng were tested for effect on TEDA assembly efficiency, and DNA usage in this region was found to be effective, optimally 200 ng;
in some implementation cases, the influence of the length of the homologous sequence between 9bp and 30bp on the assembly efficiency of TEDA is tested, and the result shows that the assembly can be completed by 9bp to 300bp, and the optimal region is 15bp to 30 bp;
in some embodiments, a linear plasmid was tested: the effect of insert molar ratio between 8:1 and 1:8 on TEDA assembly efficiency, the molar ratio between 1:1 and 1:4 was found to be the optimal region;
in some embodiments, the influence of the reaction temperature of the system in the range of 15 ℃ to 60 ℃ on the TEDA assembly efficiency is tested, and 30 ℃ is found to be the optimal reaction temperature;
in some implementation cases, the reaction time of the system is tested to be between 10min and 60min, the influence on the TEDA assembly efficiency is tested, and 40min to 60min is found to be the optimal condition;
in some embodiments, the effect of different clonotype strains on TEDA assembly efficiency was tested; the total 9 strains of DH5 alpha, JM109, Top10, XL-1 Blue MRF ', GB05, S17-1, CC118, SM10 and XL-1 Blue MRF ' are tested, and the best assembling effect of DH5 alpha and XL-1 Blue MRF ' is found;
in some embodiments, it is tested that the difference between the electric transformation, the KCM method and Inoue method for preparing competent cells for TEDA assembly is that the electric transformation cannot be used for the transformation of TEDA assembly, both KCM and Inoue can be used for the transformation of TEDA assembly, and Inoue has good assembly effect because of high transformation titer.
In some embodiments, the effect of the linear plasmid vectors with 3 'overhanging ends, 5' overhanging ends and blunt ends generated by enzyme digestion on the assembly effect is tested, and different enzyme digestion ends can complete TEDA assembly.
To further understand the kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 and the assembly technology thereof, the invention will be described in detail by citing important specific examples.
Example 1A kit for performing DNA Assembly relying on T5 exonuclease and PEG8000
The kit consists of T5 exonuclease and a buffer system containing PEG 8000; the dosage of T5 exonuclease is 0.16U/total reaction system, and the buffer system component of PEG8000 is 115mM Tris-HCl with pH 7.5, 11mM MgCl211mM DTT and 6% by mass PEG 8000.
The components in the kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 allow pre-configuration of concentration of 4/3, 2 or 5, reserve for later addition of DNA, and the concentration condition does not influence the effect of assembly reaction. T5 exonuclease and reaction buffer are mixed in the kit in advance or added later.
The kit is tested to be stored at-20 ℃ and-80 ℃ without influencing the reaction effect; and the kit can be stored at low temperature for more than half a year without influencing the reaction effect.
Example 2A kit for DNA Assembly relying on T5 exonuclease and PEG8000
The kit consists of T5 exonuclease and a buffer system containing PEG 8000; the kit is calculated by a total system of 20uL, the dosage of the T5 exonuclease is 0.04U/total reaction system, the buffer system component of PEG8000 is 105mM Tris-HCl with pH 7.5, and 10mM MgCl210mM DTT and 5% by mass of PEG 8000.
The components in the kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 allow pre-configuration of concentration of 4/3, 2 or 5, reserve for later addition of DNA, and the concentration condition does not influence the effect of assembly reaction. T5 exonuclease and reaction buffer are mixed in the kit in advance or added later.
The kit is tested to be stored at-20 ℃ and-80 ℃ without influencing the reaction effect; and the kit can be stored at low temperature for more than half a year without influencing the reaction effect.
Example 3A kit for DNA Assembly relying on T5 exonuclease and PEG8000
The kit consists of T5 exonuclease and a buffer system containing PEG 8000; the dosage of T5 exonuclease is 0.02U/total reaction system, and the buffer system component of PEG8000 is 110mM Tris-HCl with pH 7.5, 9mM MgCl29mM DTT and 4% by mass PEG 8000.
The components in the kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 allow pre-configuration of concentration of 4/3, 2 or 5, reserve for later addition of DNA, and the concentration condition does not influence the effect of assembly reaction. T5 exonuclease and reaction buffer are mixed in the kit in advance or added later.
The kit is tested to be stored at-20 ℃ and-80 ℃ without influencing the reaction effect; and the kit can be stored at low temperature for more than half a year without influencing the reaction effect.
Example 4 TEDA Assembly Single site Assembly or Multi-site Assembly
Preparation of linearized vectors and plasmids
The sequence of the template and primer required by the preparation process is referred to table 1, and the specific sequence information of the primer or fragment is referred to table 2
Table 1: obtaining inserts and linearized vectors
Table 2: sequence information of primers or fragments
The specific process is as follows:
1. the product phbCAB gene cluster is obtained by amplification of phbCAB-R and phbCAB-F primers by using the genome of Ralstonia eutropha H16 as a template (AM 260479.1). For specific primers see Table 2, the specific PCR procedure was referenced to the instructions for use of Phusion DNA polymerase (Thermol, cat # M530L). Then carrying out agarose gel electrophoresis on the PCR product, cutting the gel, recovering a target fragment, and quantifying;
2. then carrying out enzyme digestion on the product by using XhoI and Mph1103, carrying out agarose gel electrophoresis on the enzyme digestion product, cutting the gel, recovering a target fragment, and quantifying;
3. extracting p5TG plasmid (the construction method is from document Li M.et al; 2012, Microb Cell Fact), performing enzyme digestion by using XhoI and Mph1103, performing agarose gel electrophoresis on the enzyme digestion product, cutting gel, recovering target fragments, and quantifying;
4. then, ligation was carried out using T4 DNA ligase (NEB, cat # M0202S), and the concrete ligation method was performed according to the manual;
5. transforming the ligation product into escherichia coli competent cells to obtain clones, and obtaining correct clones containing p5TG:: phbCAB plasmids through enzyme digestion verification and sequencing analysis;
6. amplifying a P5tac-phbCAB fragment by taking a P5TG phbCAB plasmid as a template and using P5tac-phbCAB-Rev and P5tac-phbCAB-Fr as primers;
7. using pKD4_ Fr and pKD4_ Rev primers to expand a linearized pKD4 plasmid fragment by using pKD4 plasmid as a template;
8. carrying out In-fusion assembly reaction (Takara, cat number: 638909) on the pKD4 plasmid fragment and the P5tac-phbCAB fragment, and converting an escherichia coli XL-1 Blue MRF' strain after assembly by referring to the kit specification of a specific assembly method to obtain a pKD4, namely a P5tac-phbCAB plasmid;
9. p5tacCAB-P1-F and P5tacCAB-P1-R were used, respectively; p5tacCAB-P2-F and P5 tacCAB-P2-R; p5tacCAB-P3-F and P5 tacCAB-P3-R; p5tacCAB-P2-F and P5 tacCAB-P3-R; p5tacCAB-P1-F and P5 tacCAB-P2-R; p5tacCAB-P1-F and P5tacCAB-P3-R primer pair pKD4, P5tac-phbCAB plasmid is amplified to respectively amplify phbCAB-P1(A segment), phbCAB-P2(B segment), phbCAB-P3(C segment), phbCAB-P23(BC segment), phbCAB-P12(AB segment) and phbCAB-P123 linear segment (ABC segment);
10. after SmaI digestion is carried out on pBluescript SK- (purchased from Addgene website), SmaI-pSK linear fragments (V fragments) are obtained, 4 fragments are assembled with phbCAB-P1, phbCAB-P2 and phbCAB-P3 respectively, 3 fragments are assembled with phbCAB-P1 and phbCAB-P23, 3 fragments are assembled with phbCAB-P12 and phbCAB-P3, 2 fragments are assembled with phbCAB-P123, TEDA adopted by an assembly system is assembled, the amount of linearized plasmid is 100ng, the molar ratio of the plasmid to the inserted fragment is 1:1, the enzyme amount is 0.04U/reaction system, the temperature is 30 ℃, the reaction time is 40min, ice bath is carried out for 10min after reaction, and the total system is 20 uL.
11. After the assembly is finished, taking 5uL TEDA assembly product to convert escherichia coli cells prepared by a KCM method; after addition of LB, the culture was resumed at 37 ℃ and 250rpm for 1 hour.
12. The transformation products were spread on LB plates containing 2% glucose and 100ug/mL Amp, and the number of white colonies was counted after 20 hours.
13. The results of the experiment are shown in FIG. 2, and the method can realize assembly, and the titer of the competent cells is 1.058 multiplied by 106Under the condition of CFU/ug plasmid, for the assembly of one plasmid of about 3000bp and one linear fragment of 4417bp, 0.97X 10 can be formed in one-time performance4CFU/ug linearized plasmid. And the assembly reaction of 4 fragments can be completed, and 3.73X 10 can be obtained2CFU/ug linearized plasmid.
Example 5 PEG8000 can significantly improve the efficiency of the assembly of T5 exonuclease
Preparation of linearized vectors and plasmids
The sequences of the template, insert and linearized vector required for the preparation are referred to in Table 3, and the specific sequences of the primers or fragments are referred to in Table 4.
Table 3: obtaining inserts and linearized vectors
Sequence specific information for primers or fragments is found in Table 4.
Table 4: sequence information of primers or fragments
The specific process is as follows:
1. using pLG plasmid as a template (the construction method is shown in Li, et.al., Microb Cell fact.2012 Feb 6; 11:19.), obtaining a product eGFP through eGFP-F and eGFP-R primer amplification, then carrying out agarose gel electrophoresis on the PCR product, cutting gel, recovering a target fragment, and quantifying;
2. cutting pBluescript SK-by SmaI enzyme, carrying out agarose electrophoresis, cutting gel, recovering, carrying out 2-segment assembly with eGFP fragment, adopting TEDA assembly for an assembly system, wherein the linear plasmid dosage is 100ng, the molar ratio of the plasmid to the insert fragment is 1:1, the enzyme amount is 0.04U/reaction system, the temperature is 30 ℃, the reaction time is 40min, carrying out ice bath for 10min after reaction, and the total system is 20 uL.
3. The buffer system is provided with a sample group containing PEG8000 and a control group without PEG 8000. The buffer containing PEG8000 consisted of 100mM Tris-HCl (pH 7.5),10mM MgCl2,10mM DTT and 5% PEG 8000. The control group containing no PEG8000 had a buffer composition of 100mM Tris-HCl (pH 7.5),10mM MgCl2 and 10mM DTT.
4. Taking a TEDA assembly system configured by two different buffer systems as an assembly reaction, and taking 5uL of TEDA assembly product to convert escherichia coli cells prepared by a KCM method after the assembly reaction is finished; after addition of LB, the culture was resumed at 37 ℃ and 250rpm for 1 hour.
5. The transformed product was spread on LB plate containing 100ug/mL Amp, and the number of clones containing green fluorescence was counted after 18 hours.
6. Experimental results As shown in FIG. 3, the assembly of SmaI-pSK and eGFP can be realized by the method. And it can be seen that the group without PEG8000 formed significantly fewer positive clones than the group with PEG 8000. This indicates that PEG8000 plays an important role in the assembly system established by the present invention.
Example 6 Effect of different concentrations of T5 exouchase on Assembly experiments
Preparation of linearized vectors and plasmids
The sequence of the template and primer needed in the preparation process is referred to Table 3, and the sequence specific information is referred to Table 4.
The specific process is as follows:
1. taking pCL1920-eGFP plasmid as a template, obtaining a product eGFP through eGFP-F and eGFP-R primer amplification, then carrying out agarose gel electrophoresis on the PCR product, cutting gel, recovering a target fragment, and quantifying;
2. cutting pBluescript SK-by SmaI enzyme, carrying out agarose electrophoresis, cutting gel, recovering, carrying out 2-segment assembly with eGFP fragment, adopting TEDA assembly for an assembly system, wherein the consumption of linearized plasmid is 100ng, the molar ratio of the plasmid to the insert fragment is 1:1, the temperature is 30 ℃, the reaction time is 40min, carrying out ice bath for 10min after reaction, and the total system is 20 uL;
3. in a single TEDA assembly system of 20uL, different dosages of T5 exonuclease are introduced, and are respectively 0.02, 0.04, 0.08,0.16, 0.32 and 0.64U;
4. using TEDA assembly systems prepared by using T5 exonuclease with different amounts for assembly reaction, and taking 5uL TEDA assembly products to convert escherichia coli cells prepared by a KCM method after the assembly reaction is finished; after LB was added, the culture was resumed for 1 hour at 37 ℃ and 250 rpm;
5. the transformed product was spread on LB plate containing 100ug/mL Amp, and the number of clones containing green fluorescence was counted after 18 hours.
6. Experimental results As shown in FIG. 4, the assembly of SmaI-pSK and eGFP can be realized by the method. And it can be seen that the number of clones showed normal distribution with the increase of the amount of T5 exonuclease, and the cloning effect was between 0.02 and 0.64, but the optimum cloning efficiency was between 0.04 and 0.08 for T5 exonuclease.
Example 7 method of TEDA Assembly multiple site directed mutagenesis
Preparation of linearized vectors and plasmids
The sequences of the template, insert and linearized vector required for the preparation are referred to in Table 5, and the specific sequences of the primers or fragments are referred to in Table 6.
Table 5: obtaining inserts and linearized vectors
Table 6: sequence information of primers or fragments
The specific process is as follows:
1. the product pBBR1MCS2-linear fragment was obtained by amplification with pMCS2-F and pMCS2-R primers using pBBR1MCS-2 as template. For specific primers see Table 6, the specific PCR procedure was referenced to the instructions for use of Phusion DNA polymerase (Thermol, cat # M530L). Then carrying out agarose gel electrophoresis on the PCR product, cutting the gel, recovering a target fragment, and quantifying;
2. taking phbCAB as a template in the specification of P5TG, amplifying by using primers P5tacCAB-P1-F and P5tacCAB-P3-R to obtain a product phbCAB-P123 linear fragment, then carrying out agarose gel electrophoresis, cutting the gel, recovering a target fragment, and quantifying;
3. then TEDA is used for completing the assembly of pBBR1MCS2-linear and phbCAB-P123 linear fragments to obtain pMCS2-P5tac-phbCAB plasmid;
4. amplifying pMCS2-P5tac-phbCAB by using mut1-F and mut1-R primers to obtain a pMCS2-P5tac-phbCAB-M1-linear fragment, then carrying out agarose gel electrophoresis on a PCR product, cutting gel to recover a target fragment, then completing cyclization of a linear product by using TEDA assembly to obtain a pMCS2-P5tac-phbCAB-M1 plasmid, and introducing a TAA stop codon on a phbC gene;
5. amplifying pMCS2-P5tac-phbCAB-M1 by using mut3-F and mut3-R primers to obtain a pMCS2-P5tac-phbCAB-M13-linear fragment, then carrying out agarose gel electrophoresis on a PCR product, cutting gel to recover a target fragment, then completing self-cyclization of linear products by using TEDA assembly to obtain a pMCS2-P5tac-phbCAB-M13 plasmid, and introducing TAA stop codons on phbC and phbB genes;
6. amplifying pMCS2-P5tac-phbCAB-M13 by using mut2-F and mut2-R primers to obtain a pMCS2-P5tac-phbCAB-M123-linear fragment, then carrying out agarose gel electrophoresis on a PCR product, cutting gel to recover a target fragment, then completing self-cyclization of a linear product by using TEDA assembly to obtain a pMCS2-P5tac-phbCAB-M123 plasmid, and introducing TAA stop codons on phbC, phbA and phbB genes;
7. PCR amplification is carried out on pMCS2-P5tac-phbCAB-M123 by using a primer pair Mut1C-F, Mut3C-R, Mut1C-R, Mut2C-F, Mut2C-R and Mut3C-F respectively to obtain PMCS-P5tac-phbCAB-C1, PMCS-P5tac-phbCAB-C2 and PMCS-P5tac-phbCAB-C3 fragments; respectively using Mut1C-R and Mut3C-F to amplify pMCS2-P5tac-phbCAB-M13 plasmid to obtain a PMCS-P5tac-phbCAB-C23 fragment; using Mut1C-F and Mut1C-R to amplify pMCS2-P5tac-phbCAB-M1 to obtain a PMCS-P5tac-phbCAB-C123 fragment;
8. running agarose gel electrophoresis on the fragments recovered in the previous step and recovering by using a gel recovery kit;
9. assembling TEDA adopted by PMCS-P5tac-phbCAB-C1, PMCS-P5tac-phbCAB-C2 and PMCS-P5tac-phbCAB-C3 fragments to carry out 3 fragment assembly, wherein the consumption of linearized plasmids is 100ng, the molar ratio of the plasmids to the inserted fragments is 1:1, the enzyme amount is 0.04U/reaction system, the temperature is 30 ℃, the reaction time is 40min, ice bath is carried out for 10min after reaction, and the total system is 20 uL;
10. assembling the PMCS-P5tac-phbCAB-C1 and PMCS-P5tac-phbCAB-C23 by a similar method; meanwhile, assembling the single fragment of the PMCS-P5tac-phbCAB-C123 fragment;
11. after the assembly is finished, respectively taking 5uL TEDA assembly products to convert escherichia coli cells prepared by a KCM method; after LB was added, the culture was resumed for 1 hour at 37 ℃ and 250 rpm;
12. the transformation products were spread on LB plates containing 2% glucose and 100ug/mL Amp, and after 20 hours the number of white colonies was counted;
13. the experimental results are shown in fig. 4, the method can simultaneously realize the back mutation of 3 introduced TAA stop codons and also realize the back mutation of 1TAA stop codon.
Example 8 methods of TEDA Assembly multiple site directed mutagenesis
Preparation of linearized vectors and plasmids
The sequences of the template, insert and linearized vector required for the preparation are referred to in Table 7, and the specific sequence of the primers or fragments is referred to in Table 8.
Table 7: obtaining inserts and linearized vectors
Table 8: sequence information of primers or fragments
The specific process is as follows:
1. using pTrc99a as a template (plasmid purchased from Amersham Biosci), the product Ptrc linear fragment was obtained by amplification with the primers Ptrc-F1 and Ptrc-R1. The specific PCR procedure was described in reference to the instructions for use of Phusion DNA polymerase (Thermol, cat. No. M530L). Then carrying out agarose gel electrophoresis on the PCR product, cutting the gel, recovering a target fragment, and quantifying;
2. taking pBBR1MCS-5 as a template (plasmid is purchased from an addge website), amplifying by pMCS5-F1 and pMCS5-R1 primers to obtain a product pMCS5-linear fragment, then carrying out agarose gel electrophoresis, cutting the gel, recovering a target fragment, and quantifying;
3. then TEDA is used for completing the assembly of pMCS5-linear fragments and Ptrc linear fragments to obtain pMCS5-Ptrc plasmid;
4. pMCS5-Ptrc is amplified by using pMCS5-F2 and pMCS5-R2 primers to obtain a pMCS5-Ptrc-linear fragment, then, the PCR product is subjected to agarose gel electrophoresis, and the target fragment is recovered by cutting the gel;
5. amplifying the MG1655 genome by using LacZ-F1 and LacZ-R1 primers to obtain lacZ fragment (Gene ID:945006), then carrying out agarose gel electrophoresis on the PCR product, cutting gel and recovering the target fragment;
6. PCR amplification of pMCS5-Ptrc-LacZ with pLacZ-F and pLacZ-R primers to obtain pMCS5-Ptrc-part-LacZ-linear fragment;
7. amplifying the MG1655 genome by using LacZ-F2 and LacZ-R2 to obtain lacZ-middle-line fragment, carrying out agarose gel electrophoresis on the fragment recovered in the previous step and recovering by using a gel recovery kit;
8. concentrating TEDA assembly system mother liquor to 4/3X, 2X and 5X respectively, placing in-20 deg.C and-80 deg.C refrigerator respectively, and standing for 3 days, 10 days and 30 days respectively;
9. respectively using TEDA assembly systems which are configured into different concentration ratios, stored at different refrigerator temperatures and stored for different times for assembling pMCS5-Ptrc-part-LacZ-linear plasmid and lacZ-midle-linear insert; pMCS5-Ptrc-part-LacZ-linear plasmid and lacZ-midle-linear insert, which were not treated with TEDA assembly system, were used together as a control group; in the above experiment, the amount of linearized plasmid was 100ng, the molar ratio of plasmid to insert was 1:1, the enzyme amount was 0.04U/reaction system (excluding control), the temperature was 30 ℃, the reaction time was 40min, the reaction was followed by ice-bath for 10min, and the total system was 20 uL;
10. after the assembly is finished, respectively taking 5uL TEDA assembly products to convert escherichia coli cells prepared by a KCM method; after LB was added, the culture was resumed for 1 hour at 37 ℃ and 250 rpm;
11. the transformation products were spread on LB plates containing 2% glucose and 100ug/mL Amp, and after 20 hours the number of white colonies was counted;
12. the influence of different conditions on TEDA assembly is compared by taking the average value of the number of clones obtained by dividing the number of clones obtained by TEDA assembly under various conditions by the number of clones which can be obtained by pure DNA of the same batch without the TEDA assembly system as a comparison basis;
13. as shown in fig. 6, the test results show that the tested concentration ratio, storage temperature and storage time have no effect on the assembly effect.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shandong university
<120> kit for completing DNA assembly by relying on T5 exonuclease and PEG8000 and application thereof
<141> 2018-7-5
<160>14
<210> 1
<211> 38
<212> DNA
<213> Artificial sequence
<221>phbCAB-F
<222>（1）…（38）
<400> 1
ttaatgcatg cgaccggcaa aggcgcggca gcttccac 38
<210> 2
<211> 34
<212> DNA
<213> Artificial sequence
<221>phbCAB-R
<222>（1）…（34）
<400> 2
attctcgagt cagcccatat gcaggccgcc gttg 34
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<221> pKD4_Fr
<222>（1）…（20）
<400> 3
agccgttaag tgttcctgtg 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<221> pKD4_Rev
<222>（1）…（20）
<400> 4
atgggaatta gccatggtcc 20
<210> 5
<211> 42
<212> DNA
<213> Artificial sequence
<221>P5tac-phbCAB-Rev
<222>（1）…（42）
<400> 5
cacaggaaca cttaacggct ttatggcggg tctgctatgt gg 42
<210> 6
<211> 42
<212> DNA
<213> Artificial sequence
<221>P5tac-phbCAB-Fr
<222>（1）…（42）
<400> 6
ggaccatggc taattcccat tgctgcaagg cgattaagtt gg 42
<210> 7
<211> 45
<212> DNA
<213> Artificial sequence
<221>P5tacCAB-P1-F
<222>（1）…（45）
<400> 7
ctagaactag tggatccccc ttatggcggg tctgctatgt ggtgc 45
<210> 8
<211> 32
<212> DNA
<213> Artificial sequence
<221>P5tacCAB-P1-R
<222>（1）…（32）
<400> 8
cgagcacgtt gatcttgtcc tggccgctga tg 32
<210> 9
<211> 29
<212> DNA
<213> Artificial sequence
<221>P5tacCAB-P2-F
<222>（1）…（29）
<400> 9
ggacaagatc aacgtgctcg gcttctgcg 29
<210> 10
<211> 30
<212> DNA
<213> Artificial sequence
<221>P5tacCAB-P2-R
<222>（1）…（30）
<400> 10
gtcgacgatc atggtgtcga ccagcttggc 30
<210> 11
<211> 29
<212> DNA
<213> Artificial sequence
<221>P5tacCAB-P3-F
<222>（1）…（29）
<400> 11
tcgacaccat gatcgtcgac ggcctgtgg 29
<210> 12
<211> 47
<212> DNA
<213> Artificial sequence
<221>P5tacCAB-P3-R
<222>（1）…（47）
<400> 12
atatcgaatt cctgcagccc cagctatgag ctactcatat ggcggcc 47
<210> 13
<211> 44
<212> DNA
<213> Artificial sequence
<221>eGFP-F
<222>（1）…（44）
<400> 13
caggatgagg atcgtttcgc atgagtaaag gagaagaact tttc 44
<210> 14
<211> 44
<212> DNA
<213> Artificial sequence
<221>eGFP-R
<222>（1）…（44）
<400> 14
ttcgaacccc agagtcccgc ttatttgtat agttcataca tgcc 44
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| CN110592129A (en) * | 2019-09-16 | 2019-12-20 | 武汉赛维尔生物科技有限公司 | Novel method for constructing site-directed mutation and deletion of gene base |
| CN112063642B (en) * | 2020-09-04 | 2022-11-29 | 湖北大学 | A kind of premix and its application relying on T5 exonuclease to construct recombinant plasmid |
| CN113684202B (en) * | 2021-08-17 | 2023-08-25 | 江南大学 | A safe, non-toxic, high-throughput and seamless method for constructing plasmids |
| CN114875098B (en) * | 2022-06-29 | 2023-03-28 | 四川大学 | Kit for carrying out seamless assembly on multiple DNA fragments and assembly vector and application method thereof |
| CN115927546A (en) * | 2022-12-29 | 2023-04-07 | 北京海创科业生物科技有限责任公司 | Novel plasmid site-directed mutagenesis method based on seamless cloning technology |
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| CN106191009A (en) * | 2016-07-29 | 2016-12-07 | 苏州泓迅生物科技有限公司 | External multistage recombinase system and the application in gene assembles thereof |
| CN107384910A (en) * | 2008-02-15 | 2017-11-24 | 合成基因组公司 | The method of Ligation in vitro and combination assembling nucleic acid molecules |
| CN107760706A (en) * | 2017-10-11 | 2018-03-06 | 吴江近岸蛋白质科技有限公司 | The application of DNA excision enzymes and the method for seamless clone |
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| CN106191009A (en) * | 2016-07-29 | 2016-12-07 | 苏州泓迅生物科技有限公司 | External multistage recombinase system and the application in gene assembles thereof |
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