Expression plasmid for integrating and evolving chromosome

Abstract

The invention discloses an expression plasmid for integrating and evolving a chromosome, and the expression plasmid comprises two FRT sites, a resistance screening gene alpha, a replicon, a bacteriophage integration site (attP), a promoter (P), a multiple cloning site (MCS) behind the promoter, a terminator (TTR), a resistance screening gene beta for inducing the evolutionary process of the chromosome, and same segments A1 and A2 of two heterogenous nucleotide sequences connected with two sides of the promoter and the resistance screening gene beta, wherein the resistance screening gene alphaand the resistance screening gene beta are different resistance genes. According to the expression plasmid, a target gene can be inserted into the chromosome of engineering bacteria by virtue of simple plasmid transformation, the copy number of the target gene on the chromosome can be improved by virtue of chemical induction, the expression plasmid is simple to operate, the production efficiency of a target product can be greatly improved, and the expression plasmid is suitable for industrial production. Furthermore, the target gene is integrated onto the chromosome, so that the built engineering bacteria can not be lost in the process of passage, and is high in genetic stability and stable in production performance.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to an expression plasmid used for chromosome integration and evolution and a construction method thereof. Background technique [0002] The use of genetically engineered microorganisms to produce useful substances has become a new mode of substance production. At present, the plasmid expression system containing the target gene is usually used to construct engineered microorganisms. However, this plasmid expression system often has plasmids that are easy to lose and unstable, and the existence of plasmids will increase the metabolic burden of the host bacteria. What's more serious is that plasmids often contain antibiotic resistance selection markers, which will seriously affect environmental pollution and lead to the proliferation of antibiotic-resistant bacteria in the environment, thereby limiting the industrialization process of engineering bacteria containing plasmids...

AI Technical Summary

Problems solved by technology

Although the gene copy number of the engineered bacteria they obtained was increased due to chemically induced chromosome evolution, the gene integration technology they used was the λInCh genome integration technology, which required 4 steps, which was relatively complicated and not conducive to industrial applications, and the engineered bacteria were still Contains antibiotic resistance selection markers, not suitable for industrial production

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