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PTerm-SC plasmid as well as construction method and application thereof

A construction method and plasmid technology, which are applied in the fields of biotechnology and genetic engineering, can solve the problem that genes cannot be cloned, and achieve the effects of increasing the amount of extraction, improving stability and high stability.

Active Publication Date: 2015-04-01
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 screening function and does not have the corresponding strong promoter, other promoters in the plasmid (such as the antibiotic resistance gene promoter) can also activate the foreign gene Low-volume transcription and translation. Therefore, how to overcome the above-mentioned defects in existing plasmid vectors and improve the success rate of cloning "difficult" genes is the primary problem that many researchers need to solve

Method used

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  • PTerm-SC plasmid as well as construction method and application thereof
  • PTerm-SC plasmid as well as construction method and application thereof
  • PTerm-SC plasmid as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Construction of plasmid pTerm-SC

[0033] The structure of plasmid pTerm-SC is as follows figure 1 As shown, the construction process pattern diagram is as follows figure 2 As shown, the specific method is as follows:

[0034] 1. Design and synthesize the SopA-SopB gene. The gene sequence is shown in sequence 1 in the sequence table. The total length of the sequence is 2250bp, of which 66-1240bp is the SopA gene, which encodes the SopA protein, and 1241-2212bp is the SopB gene, which encodes the SopB protein;

[0035] 2. Design and synthesize the SopC-Terminator gene. The gene sequence is shown as sequence 2 in the sequence listing, and the total length of the sequence is 1266bp, wherein the sequence 88-542bp is the cis-acting region of SopC, and the sequence 797-1208bp is the prokaryotic transcription terminator;

[0036] 3. Design and synthesize the Cm-oriⅤ-ori2 gene. The gene sequence is shown in sequence 3 in the sequence table. The full length of t...

Embodiment 2

[0041] Example 2: Plasmid pTerm-SC Stability Detection

[0042] The plasmid pTerm-SC was transformed into Escherichia coli TOP10F 'competent cells, and single clone colonies were picked and cultivated overnight in LB medium containing chloramphenicol antibiotics (25-50 μg / μl), and then the cultured bacterial liquid was treated with Streak culture on the flat plate of chloramphenicol antibiotic (25-50 μg / μl), and re-streak culture on the plate containing chloramphenicol antibiotic (25-50 μg / μl) for the monoclonal obtained by streaking, by This resulted in pure positive transformed clones.

[0043] Inoculate the obtained pure positive transformed clones into LB medium without antibiotics, culture at 37°C for 20 passages (about 7h), and inoculate 100 μl of them into new medium LB without antibiotics, and continue culturing at 37°C 20 generations, so repeated 6 times. Finally, the bacteria of 40 generations, 60 generations, 80 generations, 100 generations and 120 generations wer...

Embodiment 3

[0046] Example 3: Plasmid pTerm-SC copy number detection

[0047] Transform pUC57 and pTerm-SC plasmids into Escherichia coli TOP10F'competent cells respectively, pick four monoclonal colonies into 4ml LB medium, culture overnight at 37°C 200rpm for 12 hours, extract the plasmids the next day, and finally dissolve the plasmids Into 50μl sterile TE, measure the concentration of the plasmid and get the concentration of pUC57: 396ng / μl, 377ng / μl, 385ng / μl, 397ng / μl, the concentration of pTerm-SC plasmid: 14.0ng / μl, 14.5ng / μl, 13.9ng / μl, 16.9ng / μl, calculate the molar concentration of pUC57 and pTerm-SC according to the measured plasmid concentration.

[0048] The molecular weights of adenine (A), guanine (G), cytosine (C), and thymine (T) are 313.2, 304.2, 329.2, and 289.2 (g / mol), respectively, then:

[0049] Molecular weight of pUC57 = 313.2 g / mol x 672 + 304.2 g / mol x 691 + 329.2 g / mol x 683 + 289.2 g / mol x 664 = 837545 g / mol;

[0050] Molecular weight of pTerm-SC = 313.2 g...

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Abstract

The invention relates to a pTerm-SC plasmid as well as a construction method and application thereof. The pTerm-SC plasmid comprises a repE gene, a sopA gene, a sopB gene, a sopC gene, an ori2 replicon, an oriV replicon, prokaryote transcription terminators and antibiotics resistance genes. The pTerm-SC plasmid has the characteristics of very low copy number, high volume and stability and the like, can be applied to gene engineering fields such as the cloning, the screening, sequencing and the genome construction of complex-structure genes including repetitive-sequence genes, instable genes and long-fragment genes and has important role in the high-efficiency and high-quality synthesis of genes.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and specifically relates to a pTerm-SC plasmid with low copy, large capacity and high stability, 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, and the size of different plasmids is between 2kb-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 oth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/10C12Q1/68
Inventor 薛高旭冯爱华孙中平廖国娟
Owner GENEWIZ INC SZ
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