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Transposase high-activity mutant in halophilic archaebacteria

A high-activity, mutant technology, applied in the field of molecular biology, can solve the problems of high price of transposase, increase of transposase purification cost, etc., achieve good tolerance and reduce purification cost

Active Publication Date: 2018-07-10
天津强微特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] During the purification process of the existing Tn5 transposase, its N-terminal sequence is easily degraded by the endogenous protease of Escherichia coli to form inactive Tn5 transposase fragments, which can also inhibit the full-length Tn5 to a certain extent. Transposase activity
In view of the above phenomenon, in the process of purifying Tn5 transposase, it is often necessary to add expensive protease inhibitors throughout the process, which increases the purification cost of transposase, and is also one of the factors for the high price of commercially available transposases

Method used

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  • Transposase high-activity mutant in halophilic archaebacteria
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  • Transposase high-activity mutant in halophilic archaebacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Construction of transposase highly active mutant Escherichia coli engineering bacteria

[0015] The highly active mutation W64K was introduced into the transposase (GenBank: KXA97509.1) gene of the non-culturable microbial halophilic archaea SCGC-AAA259I14, and the codon-optimized gene sequence was codon-optimized with reference to the codon preference of Escherichia coli. The sequence was synthesized by Jinweizhi Company and constructed into the pTYB4 expression vector to obtain the pTYB4W64K plasmid

[0016] Transform the pTYB4W64K plasmid into Escherichia coli ER2566 expression strain, use LB medium, cultivate to OD at 37 degrees 600 =0.6, add 0.1mM IPTG, lower the culture temperature to 23°C, continue to culture for 6h, and collect the cells by centrifugation.

Embodiment 2

[0017] Example 2 Purification of Transposase High Activity Mutants

[0018] Take 25-30 g of Escherichia coli cells expressing transposase highly active mutants, and add 500 mL of buffer A (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% Glycerol) at a ratio of 1:17-20 , after mixing, perform homogeneous bacteriostasis (1000bar / 2 times), after bacteriostasis, centrifuge the sample (14000 rpm, 20 min, 4°C), keep the supernatant and discard the precipitate after centrifugation.

[0019] Slowly add the centrifuged supernatant to a 5 mL Chitin column; after all the destructed supernatant passes through the Chitin column, use buffer A to wash the column for 5-7 times the column volume to ensure that some non-specific binding proteins in the column are washed out. At this time, the highly active transposase mutant is bound to the Chitin filler.

[0020] Weigh the DTT solid and dissolve it in 20 mL buffer A (the final concentration of DTT is 50 mM), slowly add the above buffer solution to the...

Embodiment 3

[0022] Example 3 Application of Transposase High Activity Mutants in the Field of Molecular Biology

[0023] Application of transposase highly active mutants in next-generation sequencing library construction:

[0024] 3.1 Construction of transposomes (Transposome)

[0025] 3.1.1 Prepare the following reaction system:

[0026]

[0027] 3.1.2. Mix the reactants evenly and react at 25°C for 30 minutes.

[0028] 3.1.3. After the reaction, the transposomes can be used for subsequent reactions immediately, or stored at -20°C.

[0029] 3.2 Fragmentation reaction (Tagmentation)

[0030] 3.2.1. Prepare the following reaction system

[0031]

[0032] 3.2.2. React at 37°C for 2 hours or at 56°C for 10-15 minutes.

[0033] 3.3 After purification, nick translation, PCR enrichment, purification and other steps, the product obtained in the previous step can be sequenced on the machine.

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Abstract

The invention belongs to the technical field of molecular biology, and particularly provides a transposase high-activity mutant derived from uncultured microorganism halophilic archaebacteria SCGC-AAA259I14. By leading the transposase high-activity mutant into escherichia coli, engineered escherichia coli conducting recombinant expression on the transposase high-activity mutant is built. The engineered escherichia coli can efficiently express the transposase high-activity mutant. Compared with the escherichia coli Tn5 transposase high-activity mutant, the recombinant transposase high-activitymutant has better stability, the purification cost can be lowered significantly, and the mutant can serve as a molecular biological reagent and is applied to the aspects such as in-vitro transpositionand second generation sequencing library building technology.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically provides a high-activity transposase mutant derived from non-culturable microorganism halophilic archaea SCGC-AAA259I14 and application thereof. Background technique [0002] Tn5 transposase is a transposase encoded by the IS50 sequence inside the complex transposon Tn5 of the IS4 family, including 476 amino acid residues and a molecular weight of 53kDa. Transposases can recognize paired end sequences (End sequences) and complete the transposition reaction by "cut and paste". [0003] The transposition reaction of transposase includes the following steps: first, Tn5 transposase recognizes the terminal sequence in the donor DNA fragment, and two Tn5 transposase molecules combine to form an active dimer; the active dimer is at the end The outside of the sequence cuts the double-stranded DNA, and cuts the end sequence and the DNA in between from the donor DNA; after tha...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12Q1/6806
CPCC12N9/1241C12Q1/6806C12Q2535/122
Inventor 王磊杨雪宁王亮郑春阳
Owner 天津强微特生物科技有限公司
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