Multi-fragment deoxyribose nucleic acid (DNA) series connection recombination assembly method based on site-specific recombination

A fragment and site technology, applied in the field of multi-fragment DNA tandem recombination and assembly, can solve problems such as unpredictable results, incompatibility of parts, and variation, and achieve the effect of a simple method.

Inactive Publication Date: 2011-12-21
FUDAN UNIV
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Problems solved by technology

[0004]The development of synthetic biology relies on basic theoretical research as a strong backing support, and understanding the basic functions of various biological components and their interactions is the premise; in addition, The innovation of technical means is also necessary, such as how to efficiently assemble complex systems together, and make it easy to replace and combine silent components? In a column in Nature, Roberta Kwok pointed out five major obstacles to the developme

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  • Multi-fragment deoxyribose nucleic acid (DNA) series connection recombination assembly method based on site-specific recombination
  • Multi-fragment deoxyribose nucleic acid (DNA) series connection recombination assembly method based on site-specific recombination
  • Multi-fragment deoxyribose nucleic acid (DNA) series connection recombination assembly method based on site-specific recombination

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[0075] 1. Construction of "entry vector":

[0076] The present invention constructs seven "entry vectors", namely pTA0006, pTA0613, pTA1307, pTA0712, pTA1203, pTA0315 and pTA1303; the first two digits of the plasmid name represent attB Mutation number, the last two digits represent attP For the mutation number, please refer to the corresponding literature [16]. The "entry vector" is constructed as follows: with primer PT1 (containing) attB 0 , SEQ No.1) and PTF (containing attP 6 , SEQ No.2) to amplify the Aparamycin resistance gene ( aac(3)IV ), the obtained PCR fragment was inserted into the pMD19-T (Takara) vector by TA cloning to obtain pTA0006 (such as figure 1 , SEQ No.3).

[0077] By the same method, other "entry vectors" were obtained: with primer PTB6 (containing attB 6 , SEQ No.4) and PTP13 (containing attP 13 , SEQ No.5) to obtain pTA0613 (SEQ No.6); with primer PTB13 (containing attB 13 , SEQ No.7) and PTP7 (containing attP 7 , SEQ No.8) to...

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Abstract

The invention belongs to the technical field of biological engineering and synthetic biology, and particularly relates to a multi-fragment deoxyribose nucleic acid (DNA) series connection recombination assembly method based on BT1 integrase and mutation recognition sites thereof. In the method, sixteen pairs of mutational integrase recognition sequences are provided through mutation screening and verification, on the basis, a complete set of related carriers is constructed and comprises a framework carrier and seven TA cloning vectors, the framework carrier can realize escherichia coli-streptomyces coelicolor shuttle, the seven TA cloning vectors comprise mutation substrates and are used for DNA element construction and screening, and a concrete method for multi-fragment external series connection recombination reaction is provided. The method of the invention is simple, fast and efficient and can be used for the multi-fragment fast series connection recombination assembly, and the amplification of series connection products is realized through escherichia coli transformation. The method can be widely used for combination and assembly of standard modules and elements in the synthetic biology.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and synthetic biology, specifically a method based on A multi-fragment DNA tandem recombination assembly method for BT1 integrase and its mutation recognition site. Background technique [0002] The term "Synthetic Biology" has been proposed for a long time. It was first seen in the 33rd volume of "Science" magazine paper in 1911, and also appeared in a book review of "The Lancet" on July 8, 1911. The word [1, 2]. After a century of rapid development of life sciences, since 2000, the term "synthetic biology" has reappeared in various academic journals and newspapers, and with the J. Mycoplasma ( Mycoplasma genitalium )[3] and Mycoplasma mycoides ( Mycoplasma mycoides ) genome[3], and successfully achieved activation in heterologous enucleated mycoplasma cells[3,4], "synthetic biology" has gradually entered the public view, and has triggered various fierce debates about the origin of l...

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/10
Inventor 丁晓明张霖赵国屏
Owner FUDAN UNIV
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