Construction method and application of pseudomonas putida suicide vector

Abstract

The invention discloses a construction method of pseudomonas putida suicide vector, as well as an application method of the suicide vector in gene knockout in pseudomonas putida. The construction method of the pseudomonas putida suicide vector comprises the following steps: replacing replicon of a pBBRlMCS-5 plasmid with R6K replicon, thereby allowing reproduction of the pBBRlMCS-5 plasmid in Escherichia coli S17 / lambda-pir while preventing reproduction of the pBBRlMCS-5 plasmid in pseudomonas putida; and then, sequentially inserting an upstream homologous arm of the target gene to be knockout, an antibiotic resistance screening gene and a downstream homologous arm of the target gene to be knockout, thereby obtaining the pseudomonas putida gene knockout suicide vector. Deletion mutant of the target gene to be knockout can be constructed by introducing the suicide vector into pseudomonas putida by performing transformation or conjugative mobilization. The construction method of the pseudomonas putida suicide vector disclosed by the invention is convenient, quick, and high in correct rate.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing a suicide vector for Pseudomonas putida and its application. Background technique [0002] Pseudomonas putida is a class of Gram-negative bacteria with extensive metabolic diversity and strong adaptability to different environments. It is often used in the study of bacterial basic metabolic pathways and is also widely used in various biotechnologies In applications such as the bioremediation of pollutants and the production of special chemical compounds, etc. Gene knockout is the main method of genetic manipulation of strains, and it is also the main method for gene and protein function research, elucidation of biodegradation pathways and regulation. Therefore, the development of efficient, fast and accurate gene knockout methods is the key to fully utilizing Pseudomonas putida. important premise. [0003] Suicide plasmids refer to some plasmids that...

AI Technical Summary

Problems solved by technology

[0004] At present, the widely used Pseudomonas putida gene knockout vector is pK18mobsacB. This traditional suicide vector has some obvious defects in gene knockout, including: (1) sacB is a lethal gene widely used as a suicide vector, but sacB The induced lethal effect of sucrose is weak in Pseudomonas putida, resulting in a very low proportion of correct deletion mutants, resulting in increased screening workload; (2) The suicide vector itself is relatively large, resulting in the efficiency of conjugative transfer of plasmids. (3) Homologous arms are connected together for two rounds of screening, but because the probability of reverting to wild type in the second round of exchange is very high, the correct rate is low, and the common method is PCR verification, which increases the screening of knockout mutants. cost

Therefore, existing suicide vectors exhibit significant limitations when used for P. putida gene knockout

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