Construction method of gene engineering strain without plasmid and antibiotic resistance screening marker

Abstract

The invention discloses a construction method of a gene engineering strain without plasmid and antibiotic resistance screening marker. A pXKF3T5b plasmid is used as the carrier to transform a target gene into a host cell, and then an auxiliary plasmid for expressing the integrase is removed, a kanamycin resistance gene in the integrated carrier is removed, the chromosome is induced by the triclosan to evolve and the evolved strain is fermented and screened, so as to obtain the gene engineering strain without plasmid but with high gene copy number; moreover, a screen marker of the pXKF3T5b plasmid is the fabI gene (for coding enoyl-acetyl carrier protein reductase) in Escherichia coli and the fabI gene is the triclosan resistance gene, thus the obtained gene engineering strain does not have the antibiotic resistance screening marker, furthermore can not cause the spread of the antibiotics drug-resistant bacteria in the environment and can be suitable for the industrialized production. In addition, the target gene of the gene engineering strain is integrated into the chromosome, thus the gene engineering strain can not be lost during sub cultures, the genetic stability is high, and the production performance is stable.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for constructing genetically engineered bacteria without plasmids and antibiotic selection markers. 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 the disadvantages that the plasmid is easily lost and unstable, and the existence of the plasmid 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 genetically ...

AI Technical Summary

Problems solved by technology

Although the gene copy number of the engineered bacteria they obtained was increased due to chemically induced chromosomal evolution, the gene integration technology they used was the λInCh genome integration technology, which required 4 steps, which was relatively complicated and the integration efficiency was low, and the engineered bacteria still contained Antibiotic resistance selection marker, not suitable for industrial production

Images