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Shuttle plasmid pSW2 based on phage as well as preparation method and application of shuttle plasmid pSW2

A technology of shuttle plasmids and phages, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc.

Active Publication Date: 2013-10-02
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, shuttle plasmids directly modified on the basis of phages are still relatively rare, and phage-based shuttle plasmids that can replicate and express in both Escherichia coli and Shewanella have not been reported yet.

Method used

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  • Shuttle plasmid pSW2 based on phage as well as preparation method and application of shuttle plasmid pSW2
  • Shuttle plasmid pSW2 based on phage as well as preparation method and application of shuttle plasmid pSW2
  • Shuttle plasmid pSW2 based on phage as well as preparation method and application of shuttle plasmid pSW2

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 , Excision of phage non-essential genes

[0044] First knock out genes that are not necessary for plasmid replication and expression in the structural unit and assembly unit of the phage, such as capsid protein, zot-like channel protein, etc., because the shuttle plasmid does not need to be packaged into active particles like a phage, the knockout The process adopts the method of double enzyme digestion, and selects AflⅡ and MluⅠ which have only one restriction site in the phage for excision.

Embodiment 2

[0045] Example 2 , Construction of replication expression plasmids shuttled between Escherichia coli and Shewanella

[0046] To successfully construct a replication expression plasmid that shuttles between E. coli and Shewanella, the following characteristics need to be obtained: (1), the plasmid must be able to replicate in E. coli and Shewanella; (2), the plasmid needs Have the gene that mediates its shuttling in order to successfully enter Shewanella from E. coli; (3), the plasmid must possess a resistance that can be selected in both E. coli and Shewanella; (4) , As an expression plasmid, it also needs a strong promoter and multiple cloning sites to accommodate and express foreign proteins;

[0047] Using different exogenous fragments (derived from different plasmids), the phage that excised the non-essential gene was modified: (1), cloning of the chloramphenicol gene: the chloramphenicol gene was derived from the pCC2FOS vector (such as figure 1 shown), the full length...

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Abstract

The invention relates to a shuttle plasmid pSW2 based on phage as well as a preparation method and an application of the shuttle plasmid pSW2. The base sequence of the shuttle plasmid is shown in SEQ ID NO.1; and a derivative plasmid is a promoter screening carrier taking a green fluorescent protein as a reporter gene. The preparation method comprises the following steps of: knocking out non-essential genes for copy expression of the plasmid out of the phage, fusing a chloramphenicol gene, cloning and replicating an initiation site, cloning a conjugal transfer component, inserting a multiple cloning site into a phage gene region, thus obtaining a pSW2 expression vector; and meanwhile, inserting the green fluorescent protein into the multiple cloning site, screening promoter sequences with different strengths, and constructing and generating the promoter screening vector. The shuttle plasmid provided by the invention can be replicated and expressed in escherichia coli and different shewanella baltica, can serve as a gene anaplerosis vector to be applied to the functional identification of shewanella baltica and can also be used for high-efficiency expression of exogenous genes at a low temperature.

Description

technical field [0001] The invention relates to a shuttle plasmid and its preparation method and application, in particular to a phage-based shuttle plasmid pSW2 and its preparation method and application. Background technique [0002] The present invention is based on the transformation of a filamentous phage derived from the deep-sea bacterium Shewanella WP3 (Shewanella piezotolerans WP3) (which has been published in "shewanella psychrophila sp.nov. and Shewanella piezotolerans sp.nov., isolated from west Pacific deep- sea ​​sediment", 2007). Bacteriophage is a type of virus. Its special feature is that it uses bacteria as its host. It is a group of genetic material wrapped in a protein shell. Most phages also have a "tail" that is used to inject genetic material into the host. The filamentous phage transformed in this patent is named SW1. Its genome is 7718bp in versatility and contains 9 possible open reading frames, encoding protein components related to its life activ...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/64
Inventor 王风平肖湘杨欣伟蹇华哗
Owner SHANGHAI JIAO TONG UNIV
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