A construction method of seamless multi-segment clone

Abstract

The invention discloses a method for constructing seamless multi-segment clones. The carrier sequentially includes a promoter sequence, a ribosome binding site, a Golden Gate reaction site, a suicide gene, a Golden Gate reaction site, a terminator, a screening gene, a large intestine Bacillus replicons and encoded repressors. This scheme has the following advantages: the vector plasmid does not need to be linearized in advance, and the amount of plasmid is small, which can reduce the cost of plasmid extraction; the restriction endonuclease digestion reaction and the ligation reaction can be carried out in the same system; each reaction is Only one restriction enzyme is needed, and there is no need to consider the conflict of reaction conditions; during the reaction, the restriction enzyme site will be excised, and there is no need to consider whether the introduction of extra fragments will have adverse effects on the target gene; by artificially controlling the sticky ends, It can avoid the background of cloning vector self-ligation; seamless connection of up to 9 fragments can be performed.

Description

technical field [0001] The present invention relates to a method for constructing a vector, in particular to a method for constructing seamless multi-segment cloning. Background technique [0002] Gene cloning, also known as molecular cloning, refers to the method of generating recombinant plasmids by linking target DNA molecule fragments with DNA molecules of specific vectors in Escherichia coli. In the traditional sense, plasmid recombination is based on the principle of base complementary pairing. The general practice is: introduce restriction sites at both ends of the target fragment through primers; use the same restriction endonuclease to digest the target fragment and plasmid vector respectively. Treatment; the target fragment and the digested product of the plasmid vector are ligated in vitro; the ligation product is transformed into Escherichia coli; the recombinant is screened. Later, in order to meet different experimental needs, on the basis of traditional plasm...

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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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