WO2017066943A1 - Method and kit for dna fragment assembling in vitro - Google Patents

Abstract

Disclosed in the present invention are a method and a kit for DNA fragment assembling in vitro, being able to assemble a plurality of DNA fragments with an assembling vector in a set fragment order. The upstream of the first fragment and the downstream of the last fragment respectively carry an overlapping sequence being same as the assembling vector. With respect to the other fragments, the upstream of each fragment carries an overlapping sequence being same as the downstream of the previous fragment, and the downstream of each fragment carries an overlapping sequence being same as the upstream of the next fragment. The inner side of the overlapping sequence of the assembling vector optionally carries restriction enzyme cutting sites respectively, and optionally carries selectable markers between the restriction enzyme cutting sites. The method of the present invention enables a plurality of DNA fragments and the assembling vector to react with DNA endonuclease, DNA exonuclease, DNA polymerase and DNA ligase, so as to accomplish the assembly.

Description

DNA fragment in vitro assembly method and kit Technical field

The invention relates to the technical field of DNA assembly, and in particular to a method and a kit for assembling a DNA fragment in vitro.

Background technique

Although DNA chemical synthesis methods can synthesize a certain length of DNA sequences from scratch, DNA synthesis of larger fragments must be completed by DNA assembly, so DNA assembly technology has become the most important basic experiment in the fields of synthetic biology and metabolic engineering. One of the technologies. The earliest DNA assembly used was the method of enzyme digestion and ligation. The method of enzyme digestion and ligation has been developed and developed for decades, and it is one of the most classic basic experimental methods in the field of genetic engineering. For example, the BioBrick method can repeatedly connect various genetic elements to a standard vector by repeating four kinds of standardized restriction enzymes, and is one of important branches of the enzyme digestion and ligation method. Another branch of the digestion and ligation method, Golden Gate, can assemble and link one or more linear or circular double-stranded DNA fragments to the assembled vector by enzymatic cleavage, ligation and simultaneous reaction. It is also a common assembly. One of the methods. However, there are still some insurmountable defects in the methods of digestion and ligation, such as restriction of restriction sites, number of fragments to be joined, and limitation of length.

With the development of synthetic biology, especially with the development of genomic artificial synthesis, the demand for large DNA fragments has increased rapidly, and the conventional methods of enzyme digestion and ligation have become inadequate. Therefore, in recent years, many new DNA assembly techniques have been developed, in which the method of assembling by using homologous sequences is the most common, forming a series of different assembly methods in vivo and in vitro, which can effectively target multiple arbitrary DNAs. The fragments are assembled to get rid of the restriction of the restriction site. In vitro assembly methods of homologous regions such as CPEC, USER, SLIC, Gibson assembly, etc., these methods can use a homologous sequence between fragments to effectively join multiple double-stranded DNA fragments, but the way to produce homologous sticky ends Different. Among them, the Gibson assembly method is especially convenient and has a wide range of applications. A double-stranded linear DNA fragment carrying a homologous region can be assembled into a double-stranded linear DNA fragment by a one-step assembly reaction. Gibson assembly also enables assembly and cloning of multiple single-stranded DNA fragments. However, the Gibson assembly one-step splicing method has high synthesis cost and low splicing efficiency. In addition, the assembly method LCL using a homologous single-stranded DNA bridge, and the yeast assembly in the yeast homologous recombination system. The method is also an effective method for DNA multi-fragment assembly, and these two methods are currently the most efficient method of assembly.

However, the existing methods are still not perfect, and higher assembly efficiency, lower error rate, simpler and faster operation process are still the pursuit of people in the development of DNA assembly technology.

Summary of the invention

The present invention provides a method for in vitro assembly of DNA fragments which depends on overlapping sequences, and can assemble and clone a plurality of DNA fragments. Accordingly, the present invention also provides kits for in vitro assembly of DNA fragments.

According to a first aspect of the present invention, the present invention provides a method for in vitro assembly of a DNA fragment, comprising the steps of assembling a plurality of DNA fragments and an assembly vector; wherein the plurality of DNA fragments are in a set fragment sequence, the first fragment The upstream and the last segment respectively have the same overlapping sequence as the above-mentioned assembled carrier, the other segments have the same overlapping sequence upstream of the previous segment, and the other segments have the same overlapping sequence upstream of the next segment; The assembled vector comprises two overlapping sequences, optionally with an enzymatic cleavage site on its inner side, optionally with a screening marker between the cleavage sites; the above method allows the plurality of DNA fragments and the above-described assembled vector to be recognized The endonuclease, exonuclease, DNA polymerase and DNA ligase of the above-mentioned cleavage site are brought into contact with each other, and the plurality of DNA fragments are assembled to the above-described assembly carrier in the set assembly order.

According to a second aspect of the present invention, there is provided a kit for in vitro assembly of a DNA fragment, comprising an assembly buffer and an assembly carrier; the kit for assembling a plurality of DNA fragments and an assembly carrier, wherein the above The DNA fragments are arranged in the same sequence of fragments, and the upstream of the first fragment and the downstream of the last fragment respectively have the same overlapping sequence as the above-mentioned assembled vector, and the other fragments have the same overlapping sequence upstream of the previous fragment, and other fragments. Downstream with the same overlapping sequence as the upstream of the next fragment; the above-described assembled vector comprises two overlapping sequences, optionally with an enzyme cleavage site on its inner side, optionally with a screening marker between the cleavage sites; Optionally, the kit further comprises at least one of a positive control test fragment, an endonuclease, an exonuclease, a DNA polymerase, and a DNA ligase.

DRAWINGS

1 is a schematic diagram showing the principle of assembly of a double-stranded circular DNA fragment according to an embodiment of the present invention;

2 is a schematic diagram showing the principle of assembling a mixed type DNA fragment according to an embodiment of the present invention;

Figure 3 is a diagram showing the assembly test of a double-stranded circular DNA fragment according to an embodiment of the present invention;

Figure 4 is a diagram showing the assembly of a double-stranded circular DNA fragment according to an embodiment of the present invention;

Figure 5 is a flat view of a mixed type DNA fragment assembly test according to an embodiment of the present invention;

Fig. 6 is a diagram showing the assembly of a mixed type DNA fragment and an enzyme digestion electrophoresis according to an embodiment of the present invention.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings.

Embodiments of the present invention describe a method of in vitro assembly of DNA fragments that rely on overlapping sequences, which can be carried out in the same reaction system to complete assembly and cloning of multiple DNA fragments.

As shown in Fig. 1, an embodiment of the present invention, for a plurality of double-stranded DNA fragments cloned on a vector (i.e., fragment 1, fragment 2, and fragment 3, in other embodiments, the number of fragments may not be limited to three) The set segment order (ie, the order of segment 1, segment 2, and segment 3 in turn), the first segment (segment 1) upstream (01) and the last segment (segment 3) downstream (02) are respectively assembled and assembled The same overlapping sequences (01 and 02) of the vector, the other fragments (segment 2) upstream (03) carry the same overlapping sequence as the downstream (03) of the previous fragment (segment 1), and the other fragments (fragment 2) are downstream (04) ) with the same overlapping sequence as the upstream (04) of the next segment (segment 3), with the overlapping sequences (01, 03, 04, 02) of each segment (segment 1, segment 2, and segment 3) The cleavage site (E); the assembled vector has two overlapping sequences (01 and 02) with an enzyme cleavage site (E) on the inside and a screening marker between the cleavage sites (Fig. 1A).

It should be noted that the fragment and vector shown in Figure 1 are both intact double-stranded circular DNA structures and thus carry an enzyme cleavage site. If the fragment and vector are linear DNA fragments, there may be no restriction sites, and thus the restriction sites are "optionally", i.e., with or without restriction sites, as appropriate. Similarly, there may or may not be a screening marker on the vector, preferably a screening marker to increase screening efficiency.

It should be noted that, in the above embodiment, although the assembled carrier has two overlapping sequences (01 and 02), it should be understood that the method of the present invention does not limit that the assembled carrier can only include two overlapping sequences, but at least Two (such as three, four or more) overlapping sequences may be present, and other overlapping sequences may be located, for example, adjacent to the screening marker.

The above plurality of DNA fragments and the assembled vector are added to the reaction system, and a DNA endonuclease, an exonuclease, a DNA polymerase, and a DNA ligase which recognize the cleavage site (E) are simultaneously added. First, the DNA endonuclease acts, and the DNA fragment and the assembled vector backbone to be assembled are excised from the plasmid (Fig. 1B), and then the DNA exonuclease acts to cleave the terminal overlapping region of the fragment and the assembled vector to reveal complementarity. The cohesive ends, while the cohesive ends are complementary paired, the fragments to be assembled and the assembled vector are sequentially ligated into a loop (Fig. 1C); then the DNA polymerase excises the remaining residue of the cleavage site and complements the remaining exonuclease The gap, finally DNA ligase acts, and the ligated open-loop plasmid is ligated into a double-stranded closed circular plasmid (Fig. 1D) and assembled. The assembly reaction is carried out in an assembly buffer that is compatible with both exonuclease, DNA polymerase, DNA ligase, and various endonucleases. The reaction product can be directly used for bacterial transformation such as Escherichia coli, and a monoclonal antibody is grown on the assembled vector resistant medium, the empty vector is removed by screening the label on the vector, the positive clone is picked, and the plasmid is obtained to obtain a large number of successfully assembled DNA. Fragment.

It should be noted that, although the method of the present invention is preferably in the assembly buffer of the same reaction system, the DNA fragment and the assembly vector are simultaneously combined with the endonuclease, exonuclease, and DNA polymerase which recognize the cleavage site. The reaction with DNA ligase is carried out, that is, a one-step reaction is preferred; however, based on the spirit of the present invention, it can also be carried out stepwise, that is, the above-mentioned DNA endonuclease, exonuclease, DNA polymerase and DNA ligase are sequentially added to the reaction. The reaction was carried out separately in the system.

As shown in Figure 2, the method of the present invention can be used not only for (a) assembly of double-stranded DNA fragments (i.e., double-stranded circular DNA fragments) which are both cloned on a vector, in which case, double-stranded DNA fragments are The outer side of the overlapping sequence has a restriction site that can be recognized by the above DNA endonuclease; and can also be used for (b) assembly of double-stranded linear DNA fragments, and (c) assembly of single-stranded linear DNA fragments. In this case, the number of single-stranded linear DNA fragments is even, and (d) any two or three types of DNA in a double-stranded DNA fragment, a double-stranded linear DNA fragment, and a single-stranded linear DNA fragment cloned on a vector a mixed assembly of fragments, in the presence of a double-stranded DNA fragment cloned on a vector, the outer side of the overlapping sequence on both sides of the double-stranded DNA fragment carries an enzyme cleavage site which is recognized by the above-mentioned DNA endonuclease; In the case of a single-stranded linear DNA fragment, the number of single-stranded linear DNA fragments is an even number. It is only necessary to have overlapping sequences as described above and as shown in FIG. The assembly principle is similar to the assembly of double-stranded circular DNA fragments.

In one embodiment of the invention, efficient assembly can be achieved with overlapping sequences greater than 15 bp in length, preferably 20-40 bp.

In one embodiment of the invention, the selection marker in the assembled vector can be any marker useful in bacteria, such as an antibiotic resistance gene, a fluorescent protein gene, a lethal gene, a lacZ gene, etc., or any combination thereof, A combination of a red fluorescent protein gene or a lacZ gene and a red fluorescent protein group gene is preferred.

In one embodiment of the present invention, the restriction site may be any restriction endonuclease recognition site or polyclonal restriction site (MCS), preferably a homeoendase (such as I-SceI) or a high density. Cloning sites (eg, GGCCGCGGCGGCGC, GTCGACCGGACCGCGGGTC, GGGCCCGCCGGCGCGCC, CGCCGGCGCGCCGCCGCCGC, GACGACGTCGACCGCGGGTC, ACCGGACCGCGGGTCACC, GGCCGCCGGCGCC, etc.).

It should be noted that the cleavage site of the assembled fragment and the assembled vector requires that the cleavage site is not present on all of the assembled fragments and the assembled vector, and the cleavage sites may be the same or different. Since the reaction is carried out in the same system, and it is required that these cleavage sites must be present on all the assembled fragments and the assembled vector, otherwise the fragments or vectors are cleaved, so the same restriction sites are preferred. In order to reduce the risk of the assembled fragments and the assembled carrier being cut, and also simple and easy to operate, the requirements for the reaction system are also the simplest.

It should be noted that all restriction enzymes can be used as the endonuclease of the present invention, and rare restriction enzymes with low probability of occurrence of cleavage sites, such as I-SceI, FseI, AscI, NotI, SfiI. , AsiSI, SbfI, RsrII, PmeI, SgrAI, TspMI, XmaI, EagI, BssHII, SacII, PacI, NaeI, NgoMIV, MluI, PspXI, AvrII, NruI, PspOMI, PvuI, BsiWI, ApaLI, PmlI, BbvCI, PshAI, AhdI , KasI, NarI, SfoI, BmtI, NheI, SalI, NmeAIII, FspI, AatII, ZraI, BglI, BlpI, AgeI, BsrBI, AleI, DraIII, Bsu36I and the like.

Furthermore, all double-stranded DNA exonucleases can be used in the present invention, preferably T5 exonuclease; all DNA polymerases can be used in the present invention, preferably DNA polymerases having 3'-exo-activity, such as phusion DNA polymerase, etc. All DNA ligases can be used in the present invention, preferably Taq DNA ligase.

In the method described in the above embodiment of the invention, the assembly vector only needs to have an overlapping sequence and an enzyme cleavage site, preferably the assembled vector described in the above embodiment of the invention. As described above, if the assembled vector is a linear DNA fragment, it may not have an enzyme cleavage site.

The invention can be used for the assembly of DNA fragments of any number and length. For double-stranded circular DNA fragments, preferably used to assemble 1-20 DNA fragments larger than 200 bp, the total length of the fragments is less than 20 kbp; For double-stranded linear DNA fragments, it is preferably used to assemble 1-10 DNA fragments larger than 200 bp, the total length of the fragments is less than 20 kbp; for single-stranded linear DNA fragments, it is preferably used to assemble 2-20 (even) DNA fragments larger than 40 nt. The total length is less than 1.5 kbp.

The process described in the above embodiment of the invention, the preferred reaction conditions are 50 ° C reaction for 1 h. If some restriction enzymes have low enzymatic activity at 50 ° C, they can be reacted at their optimum temperature (such as 37 ° C) for 0.5-1 h, and then reacted at 50 ° C for 1 h.

According to the reaction of the DNA endonuclease, exonuclease, DNA polymerase and DNA ligase of the present invention, the assembly buffer in one embodiment of the present invention contains Tris-acetic acid, magnesium ion, potassium ion, dNTPs, BSA (bovine serum albumin) and PEG 8000; preferably, the above assembly buffer further contains DTT (dithiothreitol), NAD ⁺ (nicotinamide adenine dinucleotide, coenzyme I); Change within a certain range. In a further preferred embodiment of the present invention, the assembly buffer contains Tris-acetic acid 100 mmol/L, magnesium acetate 10 mmol/L, potassium acetate 20 mmol/L, dNTPs 0.2 mmol/L, DTT 10 mmol/L, NAD ⁺ 1 mmol/ L, BSA 100 μg/mL, PEG 8000 5% (mass/volume), pH 7.5. The inventors have confirmed that the content of each of the above components varies within the range of 10% above and below the above value, and the pH is in the range of 7.2 to 7.8, and can be used as the assembly buffer of the present invention. Further, the content of each component described above is an equal-magnification relationship (for example, 2, 3, 5, 6, or 8 times) in the upper and lower 10% range of the above numerical value, and the pH may be in the range of 7.2 to 7.8, and may be configured as a mother liquid. The assembly buffer is diluted in the corresponding multiples at the time of use to obtain an assembly buffer as a working solution.

In one embodiment of the invention, a kit for in vitro assembly of DNA fragments, comprising an assembly buffer and an assembly vector, optionally further comprising a positive control test fragment, an endonuclease, an exonuclease, a DNA polymerase, and At least one of DNA ligase; the kit is used to assemble a plurality of DNA fragments and an assembly vector, wherein the plurality of DNA fragments are respectively arranged in a sequence of fragments, upstream of the first fragment and downstream of the last fragment. Assembling the same overlapping sequence of vectors, the other fragments are upstream with the same overlapping sequence as the downstream of the previous fragment, and the other fragments are downstream with the same overlapping sequence as the upstream of the next fragment; the assembled vector comprises two overlapping sequences, optionally The inside has an enzyme cleavage site. Optionally, a screening marker is placed between the cleavage sites of the above assembled vector. Optionally, the positive control test fragment described above has the same structural characteristics as the DNA fragment. Optionally, a kit may also be included in the specification, and the composition of the kit of the present invention and the method of use thereof may be described in the specification.

In a preferred embodiment of the invention, the assembly buffer and exonuclease (such as T5 DNA exo-cut) The enzyme, DNA polymerase (such as Phusion DNA polymerase) and DNA ligase (such as Taq DNA ligase) are combined into an assembled master mix. The user can carry out the assembly reaction by simply adding the DNA fragment to be assembled, the corresponding restriction enzyme and the assembly vector as required by the instructions. The assembled vector may include a plasmid having at least one antibiotic resistance such as ampicillin (Amp), kanamycin (Kan), chloramphenicol (Cm), tetracycline (Tet), and spectinomycin (Spc). The positive control test fragment can be a plurality (e.g., 3) of assembled test fragments (e.g., Saccharomyces cerevisiae synthetic sequence fragments) cloned on a vector (e.g., pMD18-T) to verify the effectiveness of the assembly system.

The DNA fragment in vitro assembly method described in the present invention can be used for gene synthesis, has few experimental steps, simple operation, and high success rate. For smaller DNA fragments, the synthesized single-stranded DNA fragments (oligos) can be directly assembled into the target fragment by one-step reaction and simultaneously cloned into the assembled vector; for larger DNA fragments or metabolic pathways, A plurality of circular plasmids carrying small DNA fragments are directly assembled into large DNA fragments by one-step assembly reaction, and simultaneously cloned into an assembly vector. It should be noted that all current assembly methods based on overlapping sequences of homologous regions cannot complete the assembly of DNA fragments cloned on the vector by one-step reaction (including assembly of multiple types of fragments mixed with DNA fragments cloned on the vector). ), and the present invention is capable of achieving this.

The DNA fragment in vitro assembly method described in the present invention can also be used for PCR product cloning and vector construction, has simple operation and high success rate, and is especially suitable for cloning and vector construction of multi-fragment, large-length PCR products. For linear PCR products, the PCR product can be cloned into an assembled vector by a one-step assembly reaction by simply introducing an overlapping sequence on the primer. Moreover, the method of the present invention not only has a higher cloning success rate for smaller PCR products (e.g., below 2.5 kbp), but also for larger PCR products (e.g., 2.5 kbp-20 kbp).

Efficient cloning For multi-fragment PCR products (if the fragment is too large to amplify well, it can be segmented by PCR) or multi-fragment vector construction, only need to introduce the fragment homologous region or overlapping sequence on the primers, still only need A single assembly reaction can assemble and clone multiple PCR products or vector fragments onto an assembly vector, thereby enabling efficient cloning of multiple fragments, which cannot be accomplished by conventional TA cloning or restriction enzyme ligation.

The DNA fragment in vitro assembly method described in the present invention can also be used for metabolic pathway optimization. Since the ribosome binding site (RBS) and some promoter sequences are short (several to several tens of bp), conventional methods are difficult to manipulate, so the method of the present invention is particularly suitable for RBS optimization and simultaneous optimization of promoter and RBS. Simply add the corresponding overlapping sequence to both ends of the RBS or promoter to be optimized, and then synthesize it in the form of single-stranded DNA and mix it into the assembly reaction, and insert it into the metabolic pathway accurately by one-step assembly.

The invention is described in detail below by way of specific examples. The experimental procedures described in the examples are only used to demonstrate the feasibility of the patented method, and the application of the inventive method is not limited thereto. The experimental procedures mentioned in the examples are routine experimental methods unless otherwise specified; the reagent consumables mentioned are conventional reagent consumables unless otherwise specified.

Example 1: Multiple cyclic DNA fragment assembly test

Taking six DNA fragments of about 1.5 kbp as an example, the feasibility of the method of the present invention for assembling a plurality of double-stranded circular DNA fragments was demonstrated. The test DNA fragments were designated as TF101, TF102, TF103, TF104, TF105 and TF106, respectively. The test fragments contained 20-40 bp homologous regions, and the upstream of TF101 and the downstream of TF106 each had a 40 bp overlapping sequence, and the restriction sites were BssHII. The six fragments are the synthetic yeast chromosome 2 sequence, and the synthetic yeast chromosome 2 sequence information can be found in the patent application 201510008356.4. The assembled vector carries the corresponding 40 bp overlapping sequence and the red fluorescent protein (mRFP1) screening marker, and the cleavage site is I-SceI.

1.1 Test DNA fragment preparation

1.1.1 PCR amplification

The test fragment TF101 was amplified using the primers TF101-F and TF101-R listed in Table 1, and the test fragment TF102 was amplified using primers TF102-F and TF102-R, and the test fragment TF103 was amplified using primers TF103-F and TF103-R. The test fragment TF104 was amplified using primers TF104-F, TF104-R, the test fragment TF105 was amplified using primers TF105-F, TF105-R, and the test fragment TF106 was amplified using primers TF106-F, TF106-R. The TF101-TF105 template was a chunkC4 plasmid (sequence is SEQ ID NO: 1), and the TF106 template was chunkD4 (sequence is SEQ ID NO: 2). The PCR reaction system was: 0.2 μL of ExTaq, 2 μL of 10×ExTaq buffer, 1.6 μL of dNTPs, 1 μL of template DNA (2 ng/μL), 1 μL of each of the positive and negative primers, and 13.2 μL of ddH ₂ O. The PCR reaction procedure was: 94 ° C for 5 min; 94 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 1.5 min, 30 cycles; 72 ° C for 5 min. The PCR product was purified using a PCR product purification kit.

Table 1

1.1.2TA clone

The PCR product was cloned into the pMD18-T vector using the TA cloning kit (TAKARA). Enzyme digestion was performed using the designed cleavage site. TF101, TF102, TF103, TF104, TF105 and TF106 plasmids were obtained, respectively.

1.2 assembly carrier preparation

The red fluorescent protein gene was amplified using the primers RFP-F, RFP-R listed in Table 1, and the amplification template was pSB2K3: J04450 (from iGEM, www.igem.org, sequence is SEQ ID NO: 17). The PCR reaction system was: 0.2 μL of Phusion DNA polymerase, 4 μL of 5×HF buffer, 1.6 μL of dNTPs, 0.6 μL of MgCl ₂ , 1 μL of template DNA (2 ng/μL), 1 μL of positive and negative primers, and ddH ₂ O 10.6 μL. . The PCR reaction procedure was: 98 ° C 30 sec; 98 ° C 10 sec, 55 ° C 30 sec, 72 ° C 1 min, 35 cycles; 72 ° C 5 min. The PCR product was purified using a PCR product purification kit.

The red fluorescent protein gene fragment and the pSBGAK vector were ligated by Gibson assembly to construct a group The carrier is named pSBGAK-RFP. The pSBGAK vector information and the Gibson assembly experimental method are described in Chinese Patent Application No. 201310085313.7.

1.3 assembly test

1.3.1 Assembly reaction

The reaction system was prepared in a 1.5 mL centrifuge tube: 2 μL of assembly buffer, 0.25 μL of Phusion DNA polymerase, 1 μL of Taq DNA ligase, 0.8 μL of 1/100 T5 exonuclease, 1 μL of BssHII, and 0.5 μL of I-SceI. 280 ng of each of TF101, TF102, TF103, TF104, TF105, and TF106 plasmids, 28 ng of pSBGAK-RFP plasmid, and 20 μL of ddH ₂ O. After reacting at 37 ° C for 30 min, the reaction was carried out at 50 ° C for 1 h.

50 μL of E. coli DH5α competent cells were transformed by heat shock method using 10 μL of the assembly reaction solution, and plated with LB medium (containing kanamycin 50 μg/mL), and cultured at 37 ° C overnight.

1.3.2 Identification of assembly results

Red and white clones were grown on the assembled transformation plates (Fig. 3), and 6 white clones (red clones were empty) were picked and inoculated into 3 mL of LB liquid medium (containing kanamycin 50 μg/mL). , shake at 37 ° C 200 rpm overnight.

Take 1.5 mL of the bacterial solution, use the plasmid small kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the plasmid, electrophoresis detection: 2 μL of plasmid DNA plus 1 μL of 10× loading buffer, mix and spot; 3 μL λ-HindIII DNA Marker (TAKARA); 1.2% agarose gel, electrophoresis at 120 V for 40 min. The plasmids of the six clones (lanes 1, 2, 3, 4, 5, 6) all met the expected size, and the results are shown in Fig. 4A.

The restriction endonucleases MluI (TAKARA) and XhoI (TAKARA) were used to identify the assembled plasmids. The total length of the assembled plasmids was 11111 bp, and two bands were generated after digestion, and the sizes were 7679 bp and 3432 bp, respectively. The digestion system was: MluI 0.3 μL, XhoI 0.3 μL, 10 μL buffer 1 μL, plasmid DNA 4 μL, and ddH ₂ O 4.5 μL. The enzyme was digested at 37 ° C for 1 h.

The results of electrophoresis detection were as follows: 1.1 μL of 10× loading buffer was added to the digested product, 6 μL of the spot was taken, 3 μL of λ-HindIII DNA Marker (TAKARA); 1.2% agarose gel, and electrophoresis at 120 V for 40 min. The digestion bands of the six plasmids (lanes 1, 2, 3, 4, 5, 6) were all in line with the expected size, and the results are shown in Figure 4B. The results of digestion of all white clones showed that the assembly accuracy of the six fragments was above 80%.

Example 2: Mixed assembly test of three types of DNA fragments

The green fluorescent protein (sfGFP) expression cassette assembly was taken as an example to demonstrate the feasibility of the method of the present invention for assembly of mixed-type DNA fragments. Wherein the promoter and terminator are cloned on the pMD18-T vector, The chain form exists, the restriction site is BssHII; the RBS sequence exists as single-stranded linear DNA (oligo); the green fluorescent protein gene fragment is a PCR product and exists as double-stranded linear DNA. The cleavage site in the assembled vector is a high-density multiple cloning site, including SfiI, BsiWI, NgoMIV, PspOMI, EagI and other cleavage sites; the screening marker is lacZ gene.

2.1 DNA fragment and assembly vector preparation

The promoters were amplified using the primers Promoter-F and Promoter-R listed in Table 2, and the terminator-F and terminator-R were used to amplify the terminator. The template was pSB2K3: J04450 plasmid. The PCR reaction system and procedure are the same as in Section 1.1.1 of the first embodiment except that the template and the primer are different. The PCR product was purified using a PCR product purification kit. The PCR product was cloned using the TA cloning kit (TAKARA). Promoter and terminator plasmids were obtained, respectively.

Table 2

The RBS sequence is a mixture of four RBSs of different intensities, which are directly synthesized by a DNA synthesizer and purified by PAGE. The RBS sequence is from iGEM (www.igem.org). The four types of RBS information are shown in Table 3.

table 3

The green fluorescent protein was amplified using the primers GFP-F and GFP-R listed in Table 4, and the template was pUC19-5_emutS_CBM_3_GFP PCR (sequence is SEQ ID NO: 30). The reaction system and procedure were the same except that the template and the primer were different. Section 1.2. The PCR product was purified using a PCR product purification kit.

The lacZ was amplified using the primers lacZ-F, lacZ-R listed in Table 4, and the template was pUC19 (Promega). The PCR reaction system and procedure are the same as in Section 1.2 of the first embodiment except that the template and the primer are different. The lacZ was purified using a PCR product purification kit and ligated into the pSBGAK vector into an assembled vector designated pSBGAK-lacZ. The assembly method is the same as the 1.2 in the first embodiment.

Table 4

2.2 assembly test

The reaction system was prepared in a 1.5 mL centrifuge tube: 10 μL of assembly buffer, 0.25 μL of Phusion DNA polymerase, 1 μL of Taq DNA ligase, 0.8 μL of 1/100 T5 exonuclease, 1 μL of BssHII, 0.5 μL of SfiI, promoter The terminator plasmid was 150 ng each, the RBS fragment was 6.25 nM, the green fluorescent protein fragment was 35 ng, the assembled vector was 28 ng, and ddH ₂ O was added to make up 20 μL. The reaction was carried out at 50 ° C for 1 h.

50 μL of E. coli DH5α competent cells were transformed by heat shock method using 10 μL of the assembly reaction solution, and plated with LB medium (containing kanamycin 50 μg/mL), and cultured at 37 ° C overnight.

2.3 Identification of assembly results

2.3.1 Enzyme digestion identification

Under blue light, it can be seen that the green clones grown on the assembled transformation plates (Fig. 5, green clones with different fluorescence intensities in the circles) were picked, and 19 clones were individually inoculated into 3 mL of LB liquid medium (including kanamycin). In 50 μg/mL), shake at 37 ° C and 200 rpm overnight.

Take 1.5 mL of the bacterial solution, use the plasmid small kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the plasmid, electrophoresis detection: 2 μL of plasmid DNA plus 1 μL of 10× loading buffer, mix and spot; 3 μL λ-HindIII DNA Marker (TAKARA); 1.5% agarose gel, electrophoresis at 180V for 30 min. The plasmids of the 19 clones all met the expected size, and the results are shown in Fig. 6A.

Two assembled plasmids were selected and identified by restriction endonuclease XhoI (TAKARA). The total length of the assembled plasmid was 3245 bp, and three bands were generated after digestion, and the sizes were 1456 bp, 1026 bp and 763 bp, respectively. The digestion system was: XhoI 0.5 μL, 1 μL of 10×H buffer, 4 μL of plasmid DNA, and 4.5 μL of ddH ₂ O. The enzyme was digested at 37 ° C for 1 h.

Electrophoresis detection results: Add 1.1 μL of 10× loading buffer to the digested product, take 6 μL spot, 3 μL D2000 DNA Marker (Tiangen Biochemical Technology (Beijing) Co., Ltd.); 1.5% agarose gel, 180V The voltage was electrophoresed for 30 min. The enzymatic cleavage bands of the two plasmids all conformed to the expected size, and the results are shown in Fig. 6B.

2.3.2 Sequencing analysis

For 19 assembled plasmids, use the primer RBSSeq-1F (sequence is SEQ ID NO: 35) for sanger Sequencing and analysis of the insertion of four RBSs in assembly. All of the 19 plasmids were sequenced and found to have good randomness. The RBS distribution is shown in Table 5.

table 5

RBS number The number of occurrences Frequency of occurrence BBa_B0034 2 10.5% BBa_B0030 6 31.6% BBa_B0032 7 36.8% BBa_B0031 4 21.1%

Example 3: Assembly kit preparation

3.1 Assembly of premixed liquid preparation

The assembly buffer of the present invention can be combined with an exonuclease (such as T5 exonuclease), a DNA polymerase (such as Phusion DNA polymerase), and a DNA ligase (such as Taq DNA ligase) into an assembled premix. The concentration of the assembly buffer may be N times (eg, 2, 3, 5, 8, or 10 times) of the working solution. The preparation of the 3× assembly buffer and the assembled premix in this example was as follows.

Prepare 3× assembly buffer; take 1 mol/L Tris-acetic acid (pH 7.5) 300 μL, 1 mol/L magnesium acetate 30 μL, 1 mol/L potassium acetate 60 μL, 10 mmol/L dNTPs 60 μL, 1 mol/ L D 30 μL, 50 mmol / L NAD ⁺ 60 μL, 10 mg / mL BSA 30 μL, PEG 8000 0.15 g, add ddH ₂ O to 1 mL, mix, and store at -20 ° C.

Prepare the assembled premix: 333 μL of 3×assembly buffer, 25 μL of Phusion DNA polymerase, 0.4 μL of T5 exonuclease, 50 μL of Taq DNA ligase, 16.6 μL of ddH ₂ O, mix and dispense into 1.5 mL centrifuge tube, 8.5 μL/tube, stored at -20 °C.

3.2 assembly carrier and test fragment preparation

The plasmid DNA of the assembled vector and the test fragment was extracted using a plasmid extraction kit. The assembled vector DNA concentration was adjusted to 28 ng/μL; the test fragment DNA concentration was adjusted to 93 ng/μL and stored at -20 °C.

3.3 Reaction system and conditions

Reaction component Amount added Assembly of premix 8.5μL Endonuclease 1μL Assembling DNA fragments * Assembly carrier 15ng

ddH2O Make up 20μL

Reaction at 50 ° C for 1 h**.

*: The molar concentration of the assembled DNA fragment was 5 times that of the assembled vector.

**: If the endonuclease activity is poor at 50 ° C, it can be reacted at 37 ° C for 30 min and then at 50 ° C for 1 h.

Reagent formula

The formulation of 1× assembly buffer was as follows: Tris-acetic acid (pH 7.5) 100 mmol/L, magnesium acetate 10 mmol/L, potassium acetate 20 mmol/L, dNTPs 0.2 mmol/L, DTT 10 mmol/L, NAD ⁺ 1 mmol/L, BSA 100 μg/mL, PEG 8000 5% (mass/volume).

The content of each component may be varied within a range of 10% above and below the above value, and the pH is in the range of 7.2 to 7.8; or the content of each component is an equal ratio multiple of the upper and lower 10% of the above value, and the pH is Within the range of 7.2 to 7.8.

The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (10)

A method for in vitro assembly of a DNA fragment, comprising the steps of assembling a plurality of DNA fragments and an assembly vector; wherein the plurality of DNA fragments are arranged in a sequence of fragments, upstream of the first fragment and downstream of the last fragment Having the same overlapping sequence as the assembled vector, the other fragments have the same overlapping sequence upstream of the previous fragment, and the other fragments have the same overlapping sequence upstream of the next fragment; the assembled vector includes two Overlapping sequences, optionally with an cleavage site on their inner side, optionally with a selection marker between the cleavage sites; said method of arranging said plurality of DNA fragments and said assembly vector and recognition site The endonuclease, exonuclease, DNA polymerase and DNA ligase of the cleavage site are contacted and reacted, and the plurality of DNA fragments are assembled to the assembly carrier in a set assembly sequence. The method according to claim 1, wherein said method comprises, in an assembly buffer of the same reaction system, said plurality of DNA fragments and said assembly vector simultaneously in the DNA identifying said restriction site The reaction is carried out by contacting with a dicer, an exonuclease, a DNA polymerase, and a DNA ligase. The method of claim 1 wherein said plurality of DNA fragments are selected from any of the following: (a) each is a double-stranded DNA fragment cloned on a vector, and the outer side of the overlapping sequence flanking the double-stranded DNA fragment respectively has an enzyme cleavage site recognizable by the endonuclease; (b) both are double-stranded linear DNA fragments; (c) each being a single-stranded linear DNA fragment, and the number of said single-stranded linear DNA fragments is an even number; or (d) including any two or three of a double-stranded DNA fragment, a double-stranded linear DNA fragment, and a single-stranded linear DNA fragment cloned on a vector; in the case where a double-stranded DNA fragment cloned on a vector is present, The outer side of the overlapping sequence flanking the double-stranded DNA fragment carries a cleavage site which can be recognized by the endonuclease; in the presence of a single-stranded linear DNA fragment, the number of the single-stranded linear DNA fragment It is even. The method according to claim 1, wherein the selection marker is selected from the group consisting of an antibiotic resistance gene, a fluorescent protein gene, a lethal gene, a lacZ gene, or any combination thereof; Optionally, the restriction site is selected from a restriction endonuclease recognition site or a polyclonal restriction site; Optionally, the endonuclease is a restriction enzyme; Optionally, the exonuclease is a T5 exonuclease; Optionally, the DNA polymerase is a pyunion DNA polymerase; Optionally, the DNA ligase is Taq DNA ligase. The method of claim 1 wherein said overlapping sequence has a length greater than 15 bp. The method according to claim 2, wherein said assembly buffer contains Tris-acetic acid, magnesium ions, potassium ions, dNTPs, BSA and PEG 8000; Optionally, the assembly buffer also contains DTT and NAD ⁺ . The method according to claim 6, wherein the assembly buffer comprises Tris-acetic acid 100 mmol/L, magnesium acetate 10 mmol/L, potassium acetate 20 mmol/L, dNTPs 0.2 mmol/L, DTT 10 mmol/L, NAD ⁺ 1 mmol/L, BSA 100 μg/mL, PEG 80005% (mass/volume), pH 7.5; or the content of each component described above varies within 10% of the above values, pH is in the range of 7.2-7.8; The content of each component is an equal-magnification relationship in the range of 10% above and below the above value, and the pH is in the range of 7.2 to 7.8. A kit for in vitro assembly of DNA fragments, comprising an assembly buffer and an assembly carrier; The kit is for assembling a plurality of DNA fragments and an assembly vector, wherein the plurality of DNA fragments are in the same sequence of fragments, and the upstream of the first fragment and the downstream of the last fragment respectively have the same as the assembled carrier Overlapping sequence, the other fragments have the same overlapping sequence upstream of the previous fragment, and the other fragments have the same overlapping sequence upstream of the next fragment; the assembled vector comprises two overlapping sequences, optionally inside Each with an enzyme cleavage site, optionally with a selection marker between the cleavage sites; Optionally, the kit further comprises at least one of a positive control test fragment, an endonuclease, an exonuclease, a DNA polymerase, and a DNA ligase. The kit according to claim 8, wherein the DNA endonuclease is a restriction enzyme, the DNA exonuclease is a T5 DNA exonuclease, and the DNA polymerase is a phusion DNA polymerase. The DNA ligase is Taq DNA ligase; Optionally, the assembly buffer is combined with the exonuclease, DNA polymerase, and DNA ligase to assemble a master mix. The kit according to any one of claims 8-9, wherein the assembly buffer contains Tris-acetic acid, magnesium ions, potassium ions, dNTPs, BSA and PEG 8000; Optionally, the assembly further comprising DTT buffer and NAD ^+; Preferably, the assembly buffer comprises Tris-acetic acid 100 mmol/L, magnesium acetate 10 mmol/L, potassium acetate 20 mmol/L, dNTPs 0.2 mmol/L, DTT 10 mmol/L, NAD ⁺ 1 mmol/L, BSA 100 μg/mL, PEG80005 % (mass/volume), pH is 7.5; or the content of each component above is within 10% of the above value, pH is in the range of 7.2 to 7.8; or the content of each component is above and below the above value The ratio is a multiple of 10%, and the pH is in the range of 7.2 to 7.8.

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