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Transformed luciferase gene, recombinant vector containing transformed luciferase gene, engineering bacterial, construction method of recombinant vector, and fermentation method of engineering bacterial

A luciferase gene and luciferase technology, applied in the field of the modified luciferase gene, can solve the problems of long breeding cycle and high breeding cost, and achieve the effects of detoxifying cells, reducing oxygen stress, and promoting expression

Inactive Publication Date: 2015-11-04
镇江泰和益元生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the limitations of current firefly luciferase production by region and season, as well as the long breeding cycle and high breeding costs, the primary purpose of the present invention is to provide a method for constructing a genetically engineered bacterium containing the firefly luciferase gene. Engineering bacteria can obtain firefly luciferase with high yield and high stability

Method used

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  • Transformed luciferase gene, recombinant vector containing transformed luciferase gene, engineering bacterial, construction method of recombinant vector, and fermentation method of engineering bacterial
  • Transformed luciferase gene, recombinant vector containing transformed luciferase gene, engineering bacterial, construction method of recombinant vector, and fermentation method of engineering bacterial
  • Transformed luciferase gene, recombinant vector containing transformed luciferase gene, engineering bacterial, construction method of recombinant vector, and fermentation method of engineering bacterial

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Embodiment 1

[0035] 1. Acquisition of firefly luciferase gene

[0036] According to the gene sequence of North American firefly luciferase, without changing its amino acid sequence, the codon was optimized to adapt to the expression system of Escherichia coli, and the gene was directly synthesized artificially (GenScript Biotech), named as Luciferase gene fl. Connect the gene with PMD-19T (TAKARA), the reaction conditions are as follows: total volume 5uL, PMD19-T vector 1uL, DNA 1uL, ddH 2 O3uL, 16°C, reaction for more than 30 minutes, sequencing (GenScript Biotech). Sequencing results show that the obtained luciferase gene fl nucleotide sequence is shown in SEQ ID NO. 1, and the recombinant vector contains luciferase gene fl, named T-fl.

[0037] 2. Firefly luciferase gene recombinant vector pET22b-fl-linker-aox

[0038] (1) Using the genomic DNA of the luciferase gene fl as a template, design primer 1 and primer 2, and obtain the PCR product fl-linker of the luciferase gene fl with a linker a...

Embodiment 2

[0050] Preparation of firefly luciferase and detection of its characteristics

[0051] 1. Obtaining firefly luciferase genetically engineered strain and inducing expression and purification of protein

[0052] (1) Acquisition of genetically engineered firefly luciferase strain

[0053] The pET22b-fl-linker-aox recombinant vector was transformed into competent cells of Escherichia coli BL21(DE3) (TAKARA), and then coated on LB solid medium containing 50mg / L of ampicillin (AMP) and cultured at 37°C. A single colony of E. coli is obtained by culturing in a box. Isolate the transformant resistant to ampicillin; extract the recombinant vector, run electrophoresis, and judge whether the pET22b-fl-linker-aox recombinant vector is successfully introduced into E. coli according to the size of the recombinant vector. If there is a fragment of about 8000bp, it means pET22b-fl-linker-aox The recombinant vector has been successfully introduced into E. coli, such as image 3 As shown, this strai...

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Abstract

The present invention belongs to the technical field of microbial fermentation, provides a transformed luciferase gene, and particularly relates to a recombinant vector containing the transformed luciferase gene, a construction method of the recombinant vector, engineering bacterial containing the recombinant vector, and a method for producing luciferase through the engineering bacterial. According to the present invention, the nucleotide sequence of the luciferase gene f1 is transformed from the firefly luciferase nucleotide sequence, and is represented by SEQ ID NO.1; the recombinant vector is pET22b-fl-linker-aox, the Rhizopus oryzae aox gene fragment is transformed into the original vector pET22b through the linker connection to obtain the recombinant vector, the vector comprises fl-linker-aox, and the sequence is represented by SEQ ID NO.2; and the preferred codons of the escherichia coli are utilized to carry out codon optimization on the firefly luciferase, such that the smooth and efficient expression of the luciferase gene in the late escherichia coli can be easily achieved.

Description

Technical field [0001] The invention belongs to the technical field of microbial fermentation, and relates to a modified luciferase gene, in particular to a recombinant vector containing the gene, a method for constructing the vector, an engineered bacteria containing the vector, and a method for the engineered bacteria to produce luciferase . Background technique [0002] Luciferase (firefly luciferase, LUC) is a general term for a class of enzymes that catalyze the oxidation and luminescence of luciferin or fatty aldehyde in organisms. Firefly luciferase is a highly efficient biological catalyst that can convert chemical energy into light energy. As early as 1884, Dubois discovered that the fluorescence disappeared quickly after grinding fireflies, and the fluorescence reappeared after adding fresh extracts from the tails of the fireflies. Later, he confirmed that there is an interaction between luciferin and luciferase in the extract, and pointed out that under the condition...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70C12N1/21C12R1/19
Inventor 黄和徐晴王磊毕建成贺沧
Owner 镇江泰和益元生物科技有限公司
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