Construction method of seamless multi-segment cloning vector

Abstract

The invention discloses a construction method of a seamless multi-segment cloning vector. The vector sequentially comprises a promoter sequence, a ribosome-binding site, a Golden Gate reaction site, asuicide gene, a Golden Gate reaction site, a terminator, a screening gene, an escherichia coli replicon and a coding suppressor. The scheme has the following advantages: plasmid of the vector is unnecessary to undergo such treatment as linear treatment and the like in advance, and the vector is low in plasmid dosage and is capable of reducing plasmid extraction cost; a restriction enzyme digestion reaction and a ligation reaction can be implemented in a same system; each reaction just needs one restriction enzyme, and such problems as reaction condition conflicts and the like cannot be takeninto consideration; in the reactions, restriction enzyme cutting sites can be excised, and it is unnecessary to consider whether the introduction of residual segments can cause bad influence to the generation of a target gene or not; through artificial control over a sticky end, a background caused on self-ligation of the cloning vector can be avoided; and seamless connection of nine segments to the greatest extent can be achieved.

Description

Technical field [0001] The invention relates to a method for constructing a vector, in particular to a method for constructing seamless multi-fragment cloning. Background technique [0002] Gene cloning, also known as molecular cloning, refers to the method of generating recombinant plasmids by linking target DNA molecular fragments with specific vector DNA molecules in E. coli. In the traditional sense, plasmid recombination is based on the principle of base complementary pairing. The general approach is: introduce restriction sites at both ends of the target fragment through primers; use the same restriction enzyme to cut the target fragment and plasmid vector separately Treatment; the target fragment and the digested product of the plasmid vector are ligated in vitro; the ligated product is transformed into E. coli; the recombinant is screened. Later, in order to meet different experimental needs, on the basis of traditional plasmid recombination technology, researchers put f...

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Problems solved by technology

[0011] In the traditional plasmid recombination scheme, restriction endonuclease digestion and ligation are carried out step by step, which takes a long time; multiple restriction endonuclease reactions also need to consider the conflict of buffer and reaction temperature; in order to obtain more Generally, the amount of plasmid required is relatively large; the problem of a large number of self-ligated vectors and a large number of background clones is common.

In order to ensure the ligation efficiency of LIC technology, the plasmid vector needs to be modified first to produce longer cohesive ends; SLIC technology and Gibson assembly rely on homologous fragments, and unsatisfactory homologous fragments are prone to produce secondary structures, etc., which affect recombination Efficiency; and in the Gibson reaction, the cohesive ends produced by restriction exonucleases for complementary base pairing are longer, and the insufficient fidelity of the polymerase in the mixed enzyme is prone to base mutation or deletion; in addition, the reaction products of Gateway technology will The introduction of redundant fragments (ie, homologous recombination sequences) into the recombinant plasmid may affect the expression of the target gene

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