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PC series plasmid as well as construction method and application thereof

A construction method and plasmid technology, which is applied in the field of biotechnology and genetic engineering, can solve the problems of gene fragment length limitation in cloning, low success rate of large fragment gene cloning, low stability of high-copy plasmids, etc., and achieve small molecular weight, high The effect of stability and large capacity

Active Publication Date: 2012-02-15
GENEWIZ INC SZ
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, the stability of high-copy plasmids is low, which limits the length of cloned gene fragments, and high-copy plasmids put a lot of pressure on host cells. Recombinant bacteria containing plasmids are usually unstable, which ultimately leads to a relatively low success rate of cloning large fragment genes. Low

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  • PC series plasmid as well as construction method and application thereof
  • PC series plasmid as well as construction method and application thereof
  • PC series plasmid as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Construction of plasmid pCA

[0048] The schematic diagram of the construction process of plasmid pCA is as follows: Figure 4 As shown, the specific method is as follows:

[0049] 1. Design and synthesize the lacZ gene, the full-length sequence is 588bp, of which 104~141 are the CAP binding region, 181~217 are the operator genes, 226~573 are the 5'-part gene sequence of the β-galactosidase gene, 243~ 323 is the multiple cloning site region. Gene sequence such as sequence 4 in the sequence listing;

[0050] 2. Design and synthesize the rop gene, the full length of the sequence is 822bp, of which the sequence 611-782 is the rop gene, which encodes the rop protein, and the gene sequence is as sequence 5 in the sequence list;

[0051] 3. Design and synthesize the rep replicon gene, with a full-length sequence of 1080bp, of which 22-689 are replicon rep genes, DNA replication start site 447 (+ / -) position, the gene sequence is as sequence 6 in the sequence ...

Embodiment 2

[0076] Embodiment 2: Construction of plasmid pCK

[0077] Change the antibiotic resistance gene on the basis of plasmid pCA to construct the plasmid pCK with kanamycin resistance, the specific method is as follows:

[0078] 1. Artificially design and synthesize the kan gene fragment, with a total length of 850 bases, of which bases 22-837 are the coding region of the kan gene. The sequence of the kan gene fragment is shown as sequence 8 in the sequence list, and the synthesized Kan electrophoresis is shown in Figure 6A , where 1 is the PCR result of the synthetic kan gene, and 2 is Marker DS5000. This figure shows that the spliced ​​gene fragment is the same as the design;

[0079] 2. Design primers P6 and P7 to amplify the part of the plasmid pCA other than the bla gene (lacZ-rop-rep). According to the designed primers, the two ends of the amplified gene fragment contain overlapping and complementary sequences with the two ends of the kan gene ;

[0080] The sequences of...

Embodiment 3

[0094] Embodiment 3: Construction of plasmid pCC

[0095] The construction method of plasmid pCC is the same as the construction method of plasmid pCK in Example 2. First, the Cmr gene is synthesized, and then primers are designed to amplify the lacZ-rop-rep gene fragment from pCA, and then the Cmr gene is reversely spliced ​​into the pCA gene The 3'-end of the fragment lacZ-rop-rep forms a linear lacZ-rop-rep-Cmr gene fragment, and finally the linear gene fragment is circularized into a circular plasmid pCC by homologous recombination.

[0096] The numbering of the bases in the plasmid pCC is the same as that of pCA and pCK. The first base T at the 5'-end of the complementary sequence of the lacZ gene fragment is used as the starting base of the plasmid sequence. The total length of the plasmid is 3338 base pairs. The sequencing results It shows that the constructed circular plasmid pCC is consistent with the expectation, and the Cmr gene is located at the position of 2539bp~...

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Abstract

The invention discloses a pC series plasmid as well as a construction method and application thereof, wherein the pC series plasmid comprises a rep gene, a rop gene, a lacZ gene and an antibiotics resistance gene which are sequentially connected with one another end to end. The pC series plasmid has the characteristics of small molecular weight, large capacity, low copy number, high stability andthe like, can be used for instable genes with complicated structures and repeated sequence, cloning, screening and sequencing long section of genes, genome construction and other gene engineering fields.

Description

technical field [0001] The invention belongs to the fields of biotechnology and genetic engineering, and specifically relates to a pC series plasmid with low copy, large capacity and high stability, its construction method and application. Background technique [0002] Plasmids are genetic components other than the chromatin of bacteria or cells that can replicate autonomously and symbiotically with bacteria or cells. It is not a necessary inheritance and case for the life activities of the host cell, but it can give the host cell some special properties, such as drug resistance, degradation or utilization of certain compounds, etc. [0003] Plasmids have the following characteristics: ①It is a double-stranded covalently closed circular DNA molecule outside the chromatin, which can naturally form a supercoiled structure, and the size of different plasmids is between 2kb and 300kb. ② It can replicate independently, and it is a replicator that can replicate independently. ③T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/65C12N15/10C12Q1/68
Inventor 柳伟强杨平刁文一杨鹏张万方孙中平廖国娟
Owner GENEWIZ INC SZ
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