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Recombinant plasmid of expression L-lysine transportprotein and engineering bacteria and application

A transport protein and recombinant plasmid technology, applied in the field of genetic engineering, can solve the problems of insufficient L-lysine transportation, etc., and achieve the effects of increasing the content of L-lysine, optimizing PCR conditions, and enhancing expression

Inactive Publication Date: 2014-04-02
SHANDONG SHOUGUANG JUNENG GOLDEN CORN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solve the problem of insufficient delivery of L-lysine in the prior art

Method used

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  • Recombinant plasmid of expression L-lysine transportprotein and engineering bacteria and application
  • Recombinant plasmid of expression L-lysine transportprotein and engineering bacteria and application
  • Recombinant plasmid of expression L-lysine transportprotein and engineering bacteria and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of L-Lysine Transporter Gene of Corynebacterium glutamicum

[0034] Pick Corynebacterium glutamicum with the preserved strain No. 1.1886 (strain alias ATCC13032) and inoculate it on the LBG solid slant medium by streaking and culturing at 30°C for 48 hours, then pick a single colony, transfer Put it in a 250ml Erlenmeyer flask containing 25ml liquid LBG medium, place it at 30°C, and cultivate it in a shaker at 100rpm until the growth of the bacteria reaches the middle of the exponential phase, about 24 hours. Then draw 5ml of bacterial liquid from this liquid LBG medium, centrifuge at 12000rpm for one minute, remove the supernatant, use the Gram-positive bacteria genome extraction kit produced by Shanghai Sangon, and extract the genome according to the instructions. Perform electrophoresis verification on a gel containing 0.5% agarose, and the extracted genome is a single band with a suitable genome size, with no serious tailing and no RNA pollution.

[0035] Sea...

Embodiment 2

[0043] Target gene expression vector construction

[0044] When designing the primers for the target gene fragment, restriction sites were designed at one end of the upstream and downstream primers. When connecting to the carrier, it is only necessary to purify the target fragment obtained by PCR and perform double digestion with EcoRI and HindIII. The vector plasmid pkk223-3 was also digested with these two restriction endonucleases, and the fragments were connected according to the general method of connecting the vector and the target fragment to construct a new expression vector. Specifically implement the enzyme digestion method, and perform double enzyme digestion on the target gene and the carrier plasmid with reference to the restriction endonucleases described in Table 2.

[0045] Table 2 restriction endonuclease double digestion reaction system

[0046]

[0047] Note: The reaction temperature is 37°C, and the enzyme produced by TAKARA company is used.

[0048] T...

Embodiment 3

[0056] Obtaining Corynebacterium glutamicum Genetic Engineering Bacteria Containing Glutamic Acid Transporter Expression Plasmid

[0057] Get the recombinant plasmid pkk-E constructed to express the L-lysine transporter, electrotransform into Corynebacterium glutamicum, and screen according to the following conditions: inoculate the transformed bacterial solution into a layer coated with ampicillin with a final concentration of On 50ug / ml LBG solid plate, culture at 30~37℃ for 30~40 hours, pick a single colony, inoculate into liquid LBG medium with a final ampicillin concentration of 50ug / ml, at 30~37℃, 30~40 Hours, 120rpm culture. Obtain genetically engineered Corynebacterium glutamicum (AT-pkk-E-DE) with doubled L-lysine transporter.

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Abstract

The invention discloses a recombinant plasmid of expression L-lysine transportprotein and engineering bacteria and application. The gene is from corynebacterium glutamicum ATCC13032, and amplified from a glutamate corynebacterium genome by polymerase chain reaction (PCR); EcoRI and HindIII restriction enzyme cutting sites are respectively added to two ends of the L-lysine transportprotein gene; pKk223-3 connection and transformation after digestion are carried out by using the same restriction enzyme; plasmids are screened and extracted, and electrically converted into the corynebacterium glutamicum ATCC13032; multiplication of the transportprotein in the corynebacterium glutamicum is realized; the molecular weight of the expression product L-lysine transportprotein is 25081.9Da. By adopting the recombinant L-lysine transportprotein or engineering bacteria of expressing the recombinant transportprotein, the L-lysine can be transferred to the outside from inside of the cell, and the recombinant plasmid has the advantages of high transport efficiency and high transfer speed, and the yield of the L-lysine is improved.

Description

[0001] technical field [0002] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant plasmid expressing an L-lysine transporter, an engineering bacterium and an application thereof. Background technique [0003] L-lysine, belonging to the aspartate family amino acids (L-aspartate family amino acids, AFAAS), including L-lysine, methionine, threonine and isoleucine 4 kinds of amino acids, all of which are essential amino acids) , is an essential amino acid for humans and animals, one of the amino acids that cannot be synthesized by themselves, and must be taken from the outside. With a variety of physiological functions, it is widely used in food, medicine and feed additives. L-Lysine is an essential building block in protein composition. As a nutrient element, the effect on the growth and development of the human body is obvious. It is also an important component in the production of carnitine, which converts part...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N1/21C12P13/08C12R1/15
Inventor 高世军刘德玉唐春辉朱艳敏
Owner SHANDONG SHOUGUANG JUNENG GOLDEN CORN CO LTD
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