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High-copy pTerm plasmid as well as construction method and application thereof

A construction method and plasmid technology, which is applied in the fields of biotechnology and genetic engineering, can solve the problem that host toxic genes cannot be cloned, and achieve the effects of large capacity, high copy number, and high stability

Active Publication Date: 2015-03-04
GENEWIZ INC SZ
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the high-copy-number plasmid with blue-white screening function provides great convenience for gene cloning, its β-galactosidase promoter is a strong promoter, which can initiate the transfer and translation of foreign genes in large quantities. This makes it impossible to clone genes with complex structures, genes whose transcription or translation products are toxic to the host

Method used

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  • High-copy pTerm plasmid as well as construction method and application thereof
  • High-copy pTerm plasmid as well as construction method and application thereof
  • High-copy pTerm plasmid as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Construction of plasmid pTerm-HA

[0039] The schematic diagram of the construction process of plasmid pTerm-HA is as follows: image 3 As shown, the specific method is as follows:

[0040] 1. Design and synthesize the bla gene with a total length of 950 bp, of which 61 to 921 are bla genes, encoding β-lactamase, resistant to ampicillin, and the gene sequence is as sequence 1 in the sequence list;

[0041] 2. Design and synthesize the colE1 replicon gene, the full-length sequence is 928bp, of which the sequence 95-777bp is the colE1 replicon gene sequence, and the gene sequence is as sequence 2 in the sequence list;

[0042] 3. Design and synthesize the Terminator gene, the full length of the sequence is 959bp, of which 51-644bp is the transcription terminator of prokaryotes and prokaryotes, and the gene sequence is as sequence 3 in the sequence list;

[0043] 4. Synthesize the above three gene fragments respectively. Since there is a 30bp overlapping regi...

Embodiment 2

[0045] Embodiment 2: Construction of plasmid pTerm-HK

[0046] The construction process of plasmid pTerm-HK is similar to the construction process of plasmid pTerm-HA, the specific method is as follows:

[0047] 1. Design and synthesize the Kan gene, the total length of the sequence is 906bp, of which bases 61-876 are the coding region of the Kan gene. Gene sequence such as sequence 4 in the sequence listing;

[0048] 2. Design and synthesize the colE1 replicon gene, the full-length sequence is 928bp, of which the sequence 95-777bp is the colE1 replicon gene sequence, and the gene sequence is as sequence 2 in the sequence list;

[0049] 3. Design and synthesize the Terminator gene, the full length of the sequence is 959bp, of which 51-644bp is the transcription terminator of prokaryotes and prokaryotes, and the gene sequence is as sequence 3 in the sequence list;

[0050] 4. Synthesize the above three gene fragments respectively. Since there is a 30bp overlapping region betw...

Embodiment 3

[0052] Example 3: Using the plasmid pTerm-HA as a template to construct the pTerm-HK plasmid

[0053] On the basis of the plasmid pTerm-HA prepared in Example 1, the plasmid pTerm-HK with kanamycin resistance was constructed by changing the antibiotic resistance gene, and the specific method was as follows:

[0054] 1. Artificially design and synthesize the Kan gene sequence, the total length of the sequence is 906bp, of which bases 61-876 are the coding region of the Kan gene. The Kan gene sequence is sequence 4 in the sequence listing;

[0055] 2. Using the pTerm-HA plasmid as a template and F-pTerm-HA / HK and R-pTerm-HA / HK as primers, the fragment colE1-Terminator was obtained by PCR.

[0056] F-pTerm-HA / HK: 5'-ctgtcagaccaagtttactcatatatactt-3';

[0057] R-pTerm-HA / HK: 5'-TTATCAGGGTTATTGTCTCATGAGCGGATA-3'.

[0058] Since the fragment has a 30 bp overlapping complementary region with both ends of the Kan gene sequence, the two fragments were assembled using the Gibson Asse...

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Abstract

The invention relates to a high-copy pTerm-series plasmid as well as a construction method and application thereof. The pTerm-series plasmid comprises a colE1 replicon gene, a procaryotic transcription terminator and an antibiotic resistance gene. The high-copy pTerm-series plasmid disclosed by the invention has the characteristics of high copy number, high capacity, high stability and the like, can be applied in the field of genetic engineering, such as cloning, screening and sequencing of complex-structure genes including repetitive sequences, instable genes and long-segment genes, constructing a genome and the like and has an important effect on high-efficiency and high-quality synthesis of genes.

Description

technical field [0001] The invention belongs to the fields of biotechnology and genetic engineering, and in particular relates to a high-copy pTerm series plasmid and its construction method and application. Background technique [0002] Plasmid is a genetic element outside chromatin, capable of autonomous replication, and is a replicator that can replicate independently. A plasmid is a double-stranded covalently closed circular DNA molecule that can naturally form a supercoiled structure. The size of different plasmids is between 2kb and 300kb. [0003] Although the plasmid is not a genetic element necessary for the survival of the host, it can impart certain special properties to the host cell, such as drug resistance. Plasmids can be used as vectors for cloning foreign genes, and are widely used in the fields of gene cloning, sequencing and gene synthesis. On the one hand, people continue to discover new plasmids from nature, and on the other hand, they also continue to...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/65
Inventor 薛高旭冯爱华孙中平廖国娟
Owner GENEWIZ INC SZ
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