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

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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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 development of current synthetic biology at the basic theoretical level [7]: (1) Many of the parts are undefined; (2) Combinations of known components The subsequent results are unpredictable (The circuitry is unpredictable); (3) The complexity of biological networks is to some extent unbearable for synthetic life (The complexity is unwieldy); (4) Many parts are incompatible with each other (Many parts are incompatible); (5) Variation may make all efforts in vain (Variability crashes the system)

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