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Novel plasmid vector and transformant capable of carrying plasmid stably

A transformant and stabilization technology, applied in the direction of using vectors to introduce foreign genetic material, botany equipment and methods, biochemical equipment and methods, etc., can solve unclear construction methods of plasmid stabilization, environmental hazards, and antibiotics to increase production costs And other issues

Active Publication Date: 2013-03-13
KANEKA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If culture is performed by applying selective pressure with antibiotics, there are problems as follows: (1) the use of antibiotic-resistant strains causes harm to the environment, (2) the amount of antibiotics necessary for culture will significantly increase production costs, (3) antibiotics Production of substances unsuitable for use in the treatment of humans and animals
However, the aforementioned stabilization of plasmids using the par region is not applicable to bacteria belonging to the genus Ralstia, Copperphyllum, and Wateria, and it is unclear whether conventional system construction methods and constructed systems are effective for plasmid stabilization. Does stabilization actually work?

Method used

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  • Novel plasmid vector and transformant capable of carrying plasmid stably
  • Novel plasmid vector and transformant capable of carrying plasmid stably
  • Novel plasmid vector and transformant capable of carrying plasmid stably

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1: Preparation of plasmid vector pCUP

[0080] The plasmid vector introduced with the replication origin region and par region in this Example is not limited as long as it can be used in bacteria belonging to the genus Ralstia. The plasmid vector prepared in this example used the replication initiation region (SEQ ID NO: 7) and the par region described in SEQ ID NO: 8 of the giant plasmid (pMOL28) possessed by C. metalloreta CH34 strain.

[0081] The specific operating procedures are as follows: first, use the DNA Purification Kit (manufactured by Promega) to prepare the DNA containing the giant plasmid from the C. metalloreta CH34 strain, and use the DNA as a template to pass through the primers described in SEQ ID NO: 1 and 2. The PCR method amplifies a DNA region of about 4 kbp containing the sequences of SEQ ID NO:7 and 8. PCR conditions are (1) 98°C, 2 minutes, (2) 98°C, 30 seconds, (3) 55°C, 30 seconds, (4) 72°C, 5 minutes, 30 cycles from (2) to (4) ...

Embodiment 2

[0083] Embodiment 2: Preparation of plasmid vector pCUP2

[0084] In order to facilitate gene introduction into the plasmid vector of the present invention, a restriction endonuclease MunI site was further introduced into pCUP obtained in Example 1. Concrete operating procedure is as follows: at first use the primer described in SEQ ID NO:5 and 6 to carry out PCR with the pCUP prepared in the embodiment 1 as template by PCR method, amplified fragment is passed DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) )) connection to introduce the MunI site to prepare pCUP2 as shown in Figure 2. PCR conditions are (1) 98°C, 2 minutes, (2) 98°C, 30 seconds, (3) 55°C, 30 seconds, (4) 72°C, 5 minutes, 30 cycles from (2) to (4) . As the polymerase, TaKaRa Pyrobest DNA Polymerase (manufactured by Takara Corporation) was used.

[0085] In this way, a plasmid vector containing the DNA region shown in SEQ ID NO: 18 and the DNA region shown in SEQ ID NO: 19 and not containing c...

Embodiment 3

[0086] Embodiment 3: Prepare transformant with plasmid vector pCUP2

[0087] Transformation was performed using electroporation as described below. The gene introduction device used for electroporation used Gene Pulser manufactured by Biorad Company, and the transformation cup was also manufactured by Biorad Company, and the gap was 0.2 cm (gap0.2 cm). Inject 400 μl of competent cells of the Ralstonia eutropha H16 strain and 5 μl of plasmid pCUP2 preparation solution into the transformation cup, put them into the pulse device, and apply electric pulses under the conditions of electrostatic capacitance 25 μF, voltage 1.5 kV, and resistance value 800 Ω. After the pulse, the bacterial solution in the transformation cup was shaken at 30° C. for 3 hours in the Nutrient Broth medium (manufactured by DIFCO), and a selection plate (Nutrient Agar medium (manufactured by DIFCO) containing 100 mg / L kanamycin ) were cultured at 30° C. for 2 days to obtain transformants.

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Abstract

It is an object of the present invention to develop a novel vector. Preferably, the object is to develop a novel vector which can be stably retained in bacteria of the genus Ralstonia, Cupriavidus or Wautersia without any antibiotic-due selective pressure and has no transferability by conjugation. Another object is to provide a strain which can stably produce polyhydroxyalkanoate using the vector, and a method for producing a polyhydroxyalkanoate using the strain. The present invention provides a novel recombinant vector which contains an origin of DNA replication functioning in bacteria of the genus Ralstonia, Cupriavidus or Wautersia . Particularly, the transformant, which is obtained by using a recombinant vector which contains the origin of DNA replication functioning in bacteria of the genus Ralstonia, Cupriavidus or Wautersia and contains a region for a recombinant vector stabilization (par region) can make the vector to be stably retained in bacteria, and can efficiently produce a polyhydroxyalkanoate.

Description

technical field [0001] The present invention relates to new vectors. In addition, the present invention belongs to the technical field of plasmid stabilization. More specifically, the present invention relates to the ability to stably exist in the genus Ralstia, Copperphyllum, or Waterella, which are hydrogen bacteria and well-known PHB-synthesizing bacteria. The recombinant vector, as well as the strain transformed by the vector and the commercial production of polyhydroxyalkanoate using the strain. Background technique [0002] When recombinant DNA technology is actually used to produce a target substance by microorganisms, there is generally a problem of instability of the recombinant plasmid. [0003] Cloning and expression vectors commonly used in laboratories are usually multi-copy plasmids, and their stable inheritance to offspring is ensured by introducing multiple plasmids into each cell genome (refer to Non-Patent Document 1). However, if a plasmid is used to int...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/00C12N1/21C12N15/09C12P7/62
CPCC12N15/74C12P7/625C12N15/52
Inventor 佐藤俊辅
Owner KANEKA CORP
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