CN115948363B - Tn5 transposase mutant and preparation method and application thereof - Google Patents
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a Tn5 transposase mutant, a preparation method and application thereof, wherein amino acids at 402 and 403 sites of Tn5 transposase are mutated, cysteine at 402 is mutated into tryptophan, glutamine at 403 is mutated into lysine, compared with wild type Tn5 transposase, the mutated Tn5 transposase has higher enzyme activity and high transposition insertion efficiency on genome DNA, and the mutated Tn5 transposase fragmentation site basically has no preference and no host pollution, thereby better meeting the requirement of high-throughput database construction.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a Tn5 transposase mutant and a preparation method and application thereof.
Background
In recent decades, NGS (Next Generation Sequencing) technology has been developed at a high speed, and sequencing instruments are constantly updated and iterated to form a large scale. In the large environment of sequencing mode industrialization, sequencing sample preparation is an important loop. Long library construction period, complicated library construction process and the like can limit the application of the NGS technology. The Transposase (Transposase) technology is introduced into the database construction to solve the problems, and the application range of the NGS is widened.
Transposases (transposases) are enzymes that perform a transposable function, usually encoded by a transposon, and recognize specific sequences of the inner end (id, ie), the outer end (oE) and the chimeric end (ME) of the transposon, which enable the transposon to be detached from adjacent sequences and inserted into a new DNA target site without homology requirements. Among them, in vitro transposition efficiency is highest with ME sequences, and Tn5 transposase is one of them, and Tn5 transposon can be inserted into the target sequence with high efficiency. The principle of Tn5 enzyme library construction specifically refers to that DNA is randomly interrupted by the transposition process of Tn5 transposomes, meanwhile, sequencing joints are inserted into two ends of fragmented DNA molecules, and finally, the construction of a DNA library containing complete joints of P5 ends and P7 ends is completed by adding sequencing indexes into PCR. The method greatly shortens the time for library construction and reduces the complex flow of the traditional library construction, so that the method is widely used in the field of second-generation sequencing at present.
However, tn5 transposase has the problems of low enzyme activity, host pollution and the like, and is greatly restricted from being applied to industrial production.
Disclosure of Invention
In view of the problems of low enzyme activity, host pollution and the like of the conventional Tn5 transposase, the invention carries out site-directed mutation on amino acid sites 402 and 403 of the Tn5 transposase (the amino acid sequence is shown as SEQ ID NO. 2), the mutated Tn5 transposase has higher enzyme activity and high transposition insertion efficiency on genome DNA, and the mutated Tn5 transposase fragmentation site is basically unbiased and free of host pollution, so that the high-flux database building requirement is better achieved.
The invention discloses a Tn5 transposase mutant, which is characterized in that amino acids at positions 402 and 403 of Tn5 transposase are mutated, cysteine at position 402 is mutated into tryptophan, glutamine at position 403 is mutated into lysine, the amino acid sequence of the mutant is shown as SEQ ID NO.2, and the nucleotide sequence of a gene for encoding the Tn5 transposase mutant is shown as SEQ ID NO. 1.
In another aspect, the invention provides recombinant plasmids containing genes encoding Tn5 transposase mutants, host cells expressing the Tn5 transposase mutants, and kits containing the Tn5 transposase mutants.
The invention also protects the application of the Tn5 transposase mutant in DNA library construction.
Compared with the prior art, the invention has the following beneficial effects:
the invention is based on rational design method, mutates amino acid of 402 and 403 sites of Tn5 transposase, the obtained mutant enzyme has more than 2 times of transposition insertion efficiency to genome DNA compared with wild Tn5 transposase, has no host pollution, and better meets the high-pass database establishment requirement.
Drawings
FIG. 1 is a diagram of the position in the tertiary structure of Tn5 transposase mutants (represented by spheres).
FIG. 2 is a graph showing the results of Tn5 transposase mutant protein purification.
FIG. 3 is a graph showing the results of the application of wild-type and mutant Tn5 transposases to the cleavage of genomic DNA.
FIG. 4 is a report of Tn5 transposase mutants after application to pooling.
FIG. 5 shows the results of data analysis of Tn5 transposase mutants after application to pooling.
Detailed Description
Example 1: preparation of mutant Tn5 transposase
1. Acquisition of mutant plasmids
The gene of the codon optimized wild Tn5 transposase is entrusted to complete gene synthesis by the biological science and technology Co., ltd, and the PTXB1 plasmid is used as a cloning vector in gene synthesis service. The constructed recombinant plasmid PTXB1-Tn5 is transferred into E.coli BL21 (DE 3) to obtain recombinant bacteria.
2. Cloning
Site-directed mutagenesis PCR was performed on C402 and Q403 of the Tn5 transposase gene locus, respectively, and primers for site-directed mutagenesis are shown in Table 1.
TABLE 1 design of site-directed mutagenesis primer in Tn5 transposase
Site-directed PCR amplification was performed using the PTXB-Tn5 plasmid as a template. The PCR amplification system was 50. Mu.L, comprising: prime STAR Max DNA Polymerase 25. Mu.L, 1. Mu.L of the upstream primer (10. Mu.M), 1. Mu.L of the downstream primer (10. Mu.M), 1. Mu.L of the plasmid template (1 ng/. Mu.L), and the total volume of the sterilized ultrapure water was made up to 50. Mu.L.
The PCR amplification procedure was: after denaturation at 98 ℃ for 1min, the amplification cycle is carried out, namely 15s denaturation at 98 ℃, 15s annealing at 55 ℃, 3min extension at 72 ℃, 28 times of total cycle and 7min extension at 72 ℃. The PCR product is detected by electrophoresis, and the band is single and clear.
The obtained fixed-point PCR reaction product is subjected to enzymolysis for 2 hours at 37 ℃ to eliminate a male parent template, the enzymolysis product is converted into chemically competent cells E.coli DH 5 alpha by a heat shock method, a LB solid plate containing ampicillin sodium (60 mug/mL) is coated with the conversion solution to obtain a fixed-point mutation library, and the fixed-point mutation library is cultured for 12 hours at 37 ℃.
3. Expression of mutant enzymes
Randomly picking 1-3 single colonies from the site-directed mutagenesis library, culturing and extracting plasmids, sample delivery to the field of the biological technology of the Optimum the nucleotide sequence was determined by the company Limited, to determine whether the expected mutation was introduced, the sequencing primer was a T7 universal primer, and the nucleotide sequence encoding the Tn5 transposase mutant is shown in SEQ ID NO. 1. Plasmid with the desired mutation was transformed into E.coli BL21 (DE 3), and single colonies were picked and inoculated into a tube with 5mL of LB liquid medium, and cultured overnight at 37℃at 200 r/min. Inoculating the cultured bacterial liquid into 100mL LB culture medium (tryptone 10g, yeast powder 5g, sodium chloride 10g, pH 7.0) containing 60 μg/mL ampicillin sodium at 1% (volume ratio), culturing at 37deg.C and 220r/min to OD 600 When the value is 0.4-0.6, adding a proper volume of IPTG (final concentration is 1 mmol/L), then carrying out induction culture for 18h at 16 ℃ and 200r/min, and collecting the thalli.
4. Expression and purification of mutant enzymes
The collected cells were washed twice with phosphate buffer, then resuspended with 10% lysis buffer (20mM Hepes,0.8M NaCl,1mM EDTA,2mM DTT,0.2%Triton X-100, 10% glycerol, naoh adjusted ph=7.2) by volume of fermentation broth, sonicated cells, and sonicated under the following conditions: the power is 300W, the operation is 3s, the interval is 6s, and the ultrasonic treatment is carried out for 8 minutes. Centrifuging the cytoblast at 10 000r/min and 4 ℃ for 30min, collecting supernatant, adding PEI with the final concentration of 0.05%, quickly and uniformly mixing on a magnetic stirrer at 4 ℃, centrifuging at high speed (10 000r/min and 30 min), collecting supernatant, and preserving at 4 ℃ to obtain Tn5 transposase crude enzyme liquid.
UsingThe pure-Cytiva protein purifier is used for purifying the protein, solves the problem of unstable batch-to-batch caused by manual operation, and comprises the following steps: after equilibration of 5mL chitin column with lysis buffer, the supernatant was passed through the chitin column at a flow rate of 0.3mL/min, the column was again washed with lysis buffer to wash away unbound materialProtein synthesis is carried out until the UV detection value shows a trend towards 0mAU; the values of UV detection showed a trend towards 40mAU by passing chitin at 2mL/min using a cleavage buffer (20mM Hepes,0.8MNaCl,1mM EDTA,100mM DTT,0.2%Triton X-100, 10% glycenol, naoh adjusted ph=7.2), the sample outlet and sample inlet were quickly placed in the same 50mL centrifuge tube and filled with 5 volumes of cleavage buffer. At this point, the UV detection value was seen to be increasing all the time by passing the flow rate, which was adjusted to 0.3mL/min, through the chitin column and cutting was performed overnight at 4 ℃. The next day the UV detection values tended to a fixed value, the cleavage buffer was collected, concentrated to 5mL using Millipore 10KDa ultrafiltration concentrate tube, and the buffer was placed in dialysis buffer (100mM Hepes,0.2M NaCl,0.2mM EDTA,2mM DTT,0.2%Triton X-100, 20% glycerol, naoh adjusted ph=7.2) and dialyzed overnight at 4 ℃. The solution after dialysis is pure enzyme, and is preserved at-80 ℃, and after the partial pure enzyme is taken out and diluted 10 times, the solution is electrophoretically detected (as shown in figure 2), and the concentration of the pure enzyme is measured.
EXAMPLE 2 use of Tn5 transposase mutants
The mutant Tn5 transposase purified by the protein purifier was subjected to the following procedure:
(1) Preparation of Tn5 transposase mutants containing a linker
To assembly buffer (50mM Hepes,0.1M NaCl,0.1mM EDTA,1mM DTT,0.1%Triton X-100, 50% glyciol, naoh adjusted ph=7.2), 2 μg of mutant Tn5 transposase obtained in example 1, 10 μΜ of the linker primer was added, reaction conditions: the Tn5 mutant transposase with the linker is obtained at 30 ℃ for 60 min.
Transposase recognition sequence: AGATGTGTATAAGAGACAG; a first linker sequence: AATGATACGGCGACACCGAGATCACTACXXXXXXTCCGGCGGCAGCGTC; a second linker sequence: caagcagaagagacggcatacgagataxxxxxxxxgtctcgtggctcgg; wherein XXXXXXXX is an Index sequence.
(2) Construction of fragmented DNA library
Preparing a treatment system with pig tissue genome DNA as a template, wherein the reaction system is as follows: 50mM TAPS,25mM MgCl 2 50% DMF, wild-type Tn5 convertants with adaptors at different input levels were used respectivelyThe enzyme loci (WT Tn5, 100 nmoL) and mutant Tn5 transposases (402 403Tn5, 25 nmoL) cleave 50ng of genomic DNA; reaction conditions: 55 ℃ for 10min. The DNA templates which are subjected to the disruption treatment of Tn5 transposase are subjected to library PCR amplification by HiFi PCR Mix forNGS (CWBIO) respectively, after amplification products are purified, library concentration measurement is carried out by using fluorescent dye Qubit, 2ng is taken for Aglient 5300 detection, and the result is shown in figure 3, when the library distribution of fragmented DNA constructed by different transposases is seen, the main peak of the library distribution obtained by mutant transposase (402 403Tn5) is approximately 232bp, and the main peak of the library distribution obtained by WT Tn5 transposase is at the position of 244, which indicates that under the condition of 50ng of genome DNA input, the effect of 100nmoL of the Tn5 transposase can be achieved by using 25nmoL of mutant Tn5 transposase, and after magnetic bead separation, the library yield can reach 240ng, thus meeting the library construction requirement, indicating that the enzyme activity of the mutant Tn5 transposase provided in example 1 is higher, and the library construction cost is greatly saved in mass library construction.
(3) GC preference analysis and host pollution of Tn5 transposase mutant
The DNA library was read using a Huada T7 sequencer for 30M data. Sequencing results are shown in FIG. 4, and GC content and distribution results show that mutant Tn5 transposase fragmentation sites are basically unbiased; the Mapped reads results in FIG. 5 indicate that the mutant Tn5 transposase is free of host contamination.
Claims (7)
- A Tn5 transposase mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 2.
- 2. A gene encoding the Tn5 transposase mutant as claimed in claim 1, wherein the nucleotide sequence of the gene is as shown in SEQ ID No. 1.
- 3. A recombinant plasmid comprising the gene of claim 2.
- 4. A host cell expressing the mutant of claim 1 or carrying the recombinant plasmid of claim 3.
- 5. Use of the Tn5 transposase mutant of claim 1 in DNA banking.
- 6. A kit comprising the Tn5 transposase mutant of claim 1.
- 7. Use of the Tn5 transposase mutant of claim 1 or the kit of claim 6 in DNA banking.
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