A kind of low copy pterm plasmid and its construction method and application

A construction method and low-copy technology, applied in the field of biotechnology and genetic engineering, can solve the problem of gene cloning and other problems, and achieve the effect of low copy number, high stability and large capacity

Active Publication Date: 2018-02-16
GENEWIZ INC SZ
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can successfully clone some "difficult" genes, there are still many genes that cannot be cloned. The reason is that although these vectors have low copy numbers, they usually have the function of blue-white screening and still have a large number of possibilities to activate foreign genes. Strong promoters for turning green and translation, and even if the plasmid does not have the blue-white sc...

Method used

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  • A kind of low copy pterm plasmid and its construction method and application
  • A kind of low copy pterm plasmid and its construction method and application
  • A kind of low copy pterm plasmid and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0040] 1. Design and synthesize the Amp gene, with a full-length sequence of 921 bp, of which 31 to 891 are the Amp gene, which encodes β-lactamase and is resistant to ampicillin. The gene sequence is as sequence 1 in the sequence list;

[0041] 2. Design and synthesize the rep replicon gene, the total length of the sequence is 971bp, of which the sequence 149-777bp is the rep replicon gene sequence, and the gene sequence is as sequence 2 in the sequence list;

[0042] 3. Design and synthesize the rop gene, the full-length sequence is 927bp, of which the sequence 561-785bp is the rop gene sequence, and the gene sequence is as sequence 3 in the sequence list;

[0043] 4. Design and synthesize the Terminator gene, with a full-length sequence of 779 bp, of which 31-289 bp are p...

Embodiment 2

[0047] Embodiment 2: Construction of plasmid pTerm-LK

[0048] The construction method of plasmid pTerm-LK is similar to that of plasmid pTerm-LA, and the specific method is as follows:

[0049] 1. Design and synthesize the Kan gene, the total length of the sequence is 876bp, of which 31-846 is the Kan gene, and the gene sequence is as sequence 5 in the sequence list;

[0050] 2. Design and synthesize the rep replicon gene, the total length of the sequence is 971bp, of which the sequence 149-777bp is the rep replicon gene sequence, and the gene sequence is as sequence 2 in the sequence list;

[0051] 3. Design and synthesize the rop gene, the full-length sequence is 927bp, of which the sequence 561-785bp is the rop gene sequence, and the gene sequence is as sequence 3 in the sequence list;

[0052] 4. Design and synthesize the Terminator gene, with a full-length sequence of 779 bp, of which 31-289 bp are prokaryotic transcription terminators, and the gene sequence is as seque...

Embodiment 3

[0056] Example 3: Using the plasmid pTerm-LA as a template to construct the pTerm-LK plasmid

[0057] On the basis of the plasmid pTerm-LA, the plasmid pTerm-LK with kanamycin resistance was constructed by changing the antibiotic resistance gene. The specific method is as follows:

[0058] The Kan gene sequence was artificially designed and synthesized, with a full length of 876bp. The gene sequence is shown as sequence 4 in the sequence list, wherein bases 31-846 are the coding region of the Kan gene.

[0059] Using the pTerm-LA plasmid prepared in Example 1 as a template, F-pTerm-LA / LK and R-pTerm-LA / LK as primers, the fragment rep-rop-Terminator was obtained by PCR;

[0060] F-pTerm-LA / LK: 5'-ctgtcagaccaagtttactcatatatactttag-3';

[0061] R-pTerm-LA / LK: 5'-actcttcctttttcaatattattgaagcatttatc-3'.

[0062] 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 Assembly Master...

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Abstract

The invention relates to a low-copy pTerm plasmid and its construction method and application. The low-copy pTerm series plasmids include rop gene, rep gene, prokaryote transcription terminator and antibiotic resistance gene. A low-copy pTerm series plasmid of the present invention has the characteristics of low copy number, large capacity, and high stability, and can be used for cloning, screening, sequencing, and genome construction of genes with complex structures, including repetitive sequences, unstable genes, and long-segment genes In the field of genetic engineering, it plays an important role in the 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 low-copy, high-capacity, high-stability pTerm 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...

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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