Oligonucleotide-linker-mediated DNA assembly method and application thereof

Abstract

The invention discloses an oligonucleotide-linker-mediated DNA assembly method and an application thereof. The assembly method comprises steps of synthesis of a long sequence, preparation and assembly of a module carrier, remolding and assembly of a skeleton carrier, preparation of a short sequence, and enzyme-cut and link-up integration of the module carrier, the remolded skeleton carrier and the short sequence. According to the method, the short sequence is a regulating element such as a promoter and an RBS, and the long sequence is a replication initiation point of a gene or a carrier. The short sequence is designed to synthesize a double-stranded DNA chain, and can connect the long sequence and be used as a barcode sequence to determine the assembly sequence in an assembly process. The gene sequence in an operon is achieved by synthesizing short sequence with different cohesive ends. According to the method, multiple factors are allowed to participate in gene regulation at the same time. The method can be used to construct a combinatorial library of a biosynthesis channel, and a high-flux optimized channel is achieved.

Description

technical field [0001] The invention belongs to the direction of synthetic biology in the field of biotechnology, relates to a DNA assembly method mediated by short-chain nucleotides and its application, in particular to the realization of high-efficiency DNA assembly technology mediated by short-chain nucleotides in metabolic engineering. A method for flux-optimizing metabolic pathways. Background technique [0002] In industry, microorganisms can be used to produce some renewable chemicals (Keasling, 2010). Today's rapid development of biotechnology, especially metabolic engineering and synthetic biology, has greatly promoted the production of microorganism-based products in the industry through regulatory pathways (Alper et al., 2005; Juminaga et al., 2012; Na et al., 2013; Smanski et al., 2014). However, in the regulation of metabolic engineering pathways, the unbalanced expression of enzymes in the pathways will lead to the accumulation of toxic intermediates, which w...

AI Technical Summary

Problems solved by technology

Therefore, limited to some extent by the ability of PCR amplification

At the same time, some unnecessary mutations are usually brought about in the PCR amplification of some longer fragments (>2kb)

Although the Golden Gate reaction can avoid mutations caused by PCR amplification, it needs to subclone each sequence into a module vector, and achieve specific sequence assembly by setting a specific barcode sequence; for multi-sequence assembly, Golden Gate The reaction needs to set different barcode sequences for subcloning for different assembly sequences, which increases human and financial costs

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