Method for quick cloning and identifying bacteria beta-galactosidase gene

A galactosidase, fast technology, applied in the fields of biotechnology and molecular biology, can solve the difficulties of cloning the β-galactosidase gene, distinguishing β-galactosidase gene fragments, and cloning the promoter sequence, etc. question

Inactive Publication Date: 2007-12-12
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the differences in β-galactosidase gene sequences from different sources, it is very difficult to use the first method to clone the β-galactosidase gene and its promoter sequence of a new species; the latter method is used to construct DNA Most of the commonly used cloning vectors for libraries use β-galactosidase as the reporter gene (such as commercial vectors pUC18, pUC19). The galactosidase gene fragments are distinguished, but cannot be used for the cloning of the β-galactosidase gene

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The method disclosed in this patent was used to clone the Bacillus megaterium ATCC14581 β-galactosidase gene.

[0034] (1)pUC18(lac - ) Vector construction

[0035] Select the existing commercial vector pUC18 that can form α-complementarity with the host strain E.coli JM109, and use its plasmid DNA as a template to amplify by reverse polymerase chain reaction (PCR). The PCR product was recovered by cutting gel, and then treated with Xho I endonuclease at 37°C for 4 hours. The digested fragment was purified by a DNA recovery kit (product of OMEGA), and then T was added. 4 DNA ligase was ligated at 16°C for 10 hours. Take 10μL of ligation solution and mix with 100μL of E.coli JM109 competent cells, ice bath for 30min, heat shock at 42℃ for 90s, put on ice for 2min, add 500μL of LB medium, incubate at 37℃ for 2h, and apply 100μg / mL ampicillin LB medium plate, cultivated overnight at 37°C. Pick the transformants and extract the plasmid DNA to obtain the genetically modified vec...

Embodiment 2

[0056] The method disclosed in this patent was used to clone the Bacillus megaterium ATCC14581 β-galactosidase gene.

[0057] (1)pUC19(lac - ) Vector construction

[0058]Select the existing commercial vector pUC19 that can form α-complementarity with the host strain E.coli JM109, and use its plasmid DNA as a template to amplify by reverse polymerase chain reaction (PCR). The latter PCR products were recovered by cutting gel, and then treated with Xho I endonuclease at 37°C for 4 hours. The digested fragments were purified by DNA recovery kit (product of OMEGA), and then added T 4 DNA ligase was ligated at 16°C for 10 hours. Take 10μL ligation solution and mix with 100μL E.coli JM109 competent cells, ice bath for 30min, heat shock at 42℃ for 90s, put on ice for 2min, add 500μL LB medium, incubate at 37℃ for 2h, and spread 100μg / mL ampicillin LB medium plate, cultivated overnight at 37°C. Pick the transformants and extract the plasmid DNA to obtain the genetically modified vector p...

Embodiment 3

[0079] The method disclosed in this patent was used to clone the Bacillus circulans ATCC 31382 β-galactosidase gene.

[0080] (1)pUC18(lac - ) Vector construction

[0081] Select the existing commercial vector pUC18, which can form α-complementarity with the host bacteria E.coli DH5α, and use its plasmid DNA as a template to amplify by reverse polymerase chain reaction (PCR). The latter PCR products were recovered by cutting gel, and then treated with Xho I endonuclease at 37°C for 4 hours. The digested fragments were purified by DNA recovery kit (product of OMEGA), and then added T 4 DNA ligase was ligated at 16°C for 10 hours. Take 10μL of ligation solution and mix with 100μL of E.coli DH5α competent cells, ice bath for 30min, heat shock at 42°C for 90s, place on ice for 2min, add 500μL LB medium, incubate at 37°C for 2 hours, and coat with 100μg / mL ampicillin Penicillin LB medium plate, 37 ℃ culture overnight. Pick the transformants and extract the plasmid DNA to obtain the gen...

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Abstract

The invention discloses a method for coloning and determining beta- galactosidase gene. The invention deletes nucleotide squence carried on vector, at 334- 489 position with its length being 156bp by using adverse PCR technique based on vector pUC18 and pUC19, and produces vector pUC18 (lac-) and pUC19 (lac-). The vector pUC18 (lac-) and pUC19 (lac-) are used to construct DNA database, and determines and clone recon that contains beta- galactosidase gene according to the blue or white bacteria group formed on LB culture medium plate of IPTG and X-gal, and determines promotor type for beta- galactosidase gene according to if IPTG induction is necessary for blue bacteria group formation.

Description

Technical field [0001] The invention belongs to the fields of biotechnology and molecular biology, and particularly relates to a method for cloning and identifying bacterial β-galactosidase genes. The method lays a foundation for quickly obtaining β-galactosidase genes. Background technique [0002] β-galactosidase (β-galactosidase), trade name lactase (lactase, Lac), can hydrolyze lactose to produce galactose and glucose, making lactose in milk easy to absorb. β-galactosidase is widely distributed in animals, plants and microorganisms (Gao Huanchun, 1996), but for humans, with age, the activity of lactase in the intestine decreases rapidly, causing "lactose intolerance"; in lactic acid bacteria cells Generally, it has β-galactosidase activity. The lactose content of dairy products fermented by lactic acid bacteria is greatly reduced (de Vrese M, et al, 2001). The addition of exogenous lactase is also an effective way to reduce lactose in dairy products (Wang Guoyu, 1999) ). At p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/70C12N1/21
Inventor 孔健孔文涛季明杰
Owner SHANDONG UNIV
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