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Seamless DNA assembling method being high in efficiency and low in cost

A low-cost, high-efficiency technology, applied in the field of bioengineering, can solve problems such as cumbersome operation procedures and expensive kits

Inactive Publication Date: 2019-12-20
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods all require in vitro ligation of linear DNA fragments before being transferred into host cells, and the operation process is cumbersome; in addition, most of the commercial kits are expensive, for example: NEBuilder kit launched by NEB Company averages becomes $18.5

Method used

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  • Seamless DNA assembling method being high in efficiency and low in cost
  • Seamless DNA assembling method being high in efficiency and low in cost

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[0024] (1) Production of DH10B*transformation competent cells: ①First pick a single colony from the DH10B* plate, put it into 5ml of liquid LB medium and culture it overnight in a shaker at 37°C; ②From the culture Take 50μl from the solution and add it to a new 50ml liquid medium and culture it at 37℃ to OD 600 When it reaches 0.3, add 80 μl of IPTG with a concentration of 100 mM, and then put it in a shaker to continue culturing until OD 600 reach 0.5-0.6; ③ Shake the 50ml bacterial solution, put it in an ice bath for 30 minutes, and turn on the centrifuge at the same time and adjust it to 4°C for pre-cooling; ④ After 30 minutes (all operations are carried out at 4°C), transfer the bacterial solution into the centrifuge in the ultra-clean bench tube, and centrifuge at 5000rpm at 4°C for 2min to precipitate the bacteria; ⑤Pour off the supernatant and add 22.5ml 0.1M Cacl 2 After resuspension, let stand for 5-10min, then centrifuge at 5000rpm, 4°C for 2min to precipitate the b...

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Abstract

A method provided by the invention comprises the steps of performing engineering reforming on escherichia coli DH10B, knocking out recB-recC-rec-D genes on a genome, and performing overexpression on recE-recT genes on the genome to obtain a recombinant engineering host bacterial strain DH10B* being good in performance; and co-transferring 1-3 DNA fragments with 20bp-40bp homologous sequence jointsinto cells to realize in vivo assembly of a complete plasmid, wherein the positive rate of colonies formed on a flat is higher than 95%. Compared with a conventional seamless DNA assembling method, the method disclosed by the invention omits expensive and time-consuming in vitro connection operation, realizes high-efficiency, low-cost and low-time-consumption seamless DNA assembling, and has great application value in molecular biology experiments.

Description

technical field [0001] The invention relates to the genetic engineering transformation of Escherichia coli DH10B strain and the optimization of seamless DNA assembly process, belonging to the technical field of bioengineering. Background technique [0002] DNA assembly technology is an indispensable link in molecular biology, metabolic engineering and synthetic biology. The traditional enzyme-cut ligation method has been gradually eliminated due to the restriction of the enzyme-cutting site. The emergence of seamless DNA assembly technology breaks the restriction of the enzyme-cutting site, making it possible to assemble arbitrary DNA fragments. The difference between seamless DNA assembly and enzyme-cut ligation is that the ends of the linear DNA fragments to be ligated have homologous sequences, and the fragments rely on the complementary pairing of homologous sequences to form a circle. In recent years, more and more seamless DNA assembly methods have been reported, and ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70
CPCC12N15/70
Inventor 霍毅欣王雪芹黄潮勇
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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