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Pseudomonas putida gene knockout and genome simplification system

A Pseudomonas putida gene knockout technology, applied in the field of microbial genetic engineering, can solve the problems of high economic cost, troublesome knockout screening, low accuracy rate, etc., achieve simple and efficient operation, increase the probability of correct mutation, and correct rate-enhancing effect

Active Publication Date: 2016-11-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the widely used Pseudomonas putida knockout vector is pK18mobsacB. In the traditional knockout plasmid construction process, the homology arms are connected together for a second round of selection, but the probability of returning to the wild type due to the second round of exchange is very high. Large, it brings a lot of trouble for knockout screening, the correct rate is very low, and the second round of screening can only be verified by PCR, resulting in high economic costs
This traditional suicide plasmid knockout approach is not applicable for genome reduction in Pseudomonas putida

Method used

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  • Pseudomonas putida gene knockout and genome simplification system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of template plasmid pWJW101 and expression plasmids pWJW102 and pWJW103

[0031] (1) Construction of the template plasmid pWJW101 for amplifying the Gm-resistant fragment with loxL / R sites in the same direction at both ends: the first step: using pBBR1MCS-5 (Kovach, M.E. et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS,carrying different antibiotic-resistance cassettes.Gene 166,175-176(1995).) as a template, gm-F / gm-R primer pair as a template, PCR amplification to obtain the gm gene, and in its SmaI restriction sites were introduced at both ends, after enzyme digestion and purification, it was ready for use; pDTW202 (Hu J, TanY, LiY, Hu X, Xu D, Wang X. Construction and application of an efficient multiple-gene-deletion system in Corynebacterium glutamicum.Plasmid, 2013,70 (3): 303-313) plasmid as template, with pBSloxL-F and pBSloxR-R primers as template, PCR amplification obtains the linear pBSloxL / R plasmid fragment ...

Embodiment 2

[0043] Example 2 Knockout of PP5288 in Pseudomonas putida KT2442

[0044] 1. Obtaining the knockout plasmid pWJW201

[0045] Using the genome of Pseudomonas putidaKT2442 as a template, the upstream and downstream fragments of the PP5288 gene were amplified with corresponding primers, respectively. EcoRI and XbaI were introduced at the 5' and 3' ends of the upstream fragment, respectively, and BamHI and HindIII restriction sites were introduced at the 5' and 3' ends of the downstream fragment, respectively. Using pWJW101 as a template and primers

[0046] gm-lox-XbaI(+)acctctagaAATACGACTCACTATAGGGCG,

[0047] gm-lox-BamHI(-)cgggatccGCGCAATTAACCCTCACTAAAG,

[0048] The gm cassette containing loxL / R sites at both ends was amplified, and XbaI and BamHI restriction sites were introduced at the 5' and 3' ends, respectively. The upstream fragment of PP5288 was purified by EcoRI and XbaI digestion, the downstream fragment was purified by BamHI and HindIII digestion, the gm box fra...

Embodiment 3

[0082] Knockout of gene cluster PP3126-PP3142 in Pseudomonas putida KT2442 in embodiment 3

[0083] 1. Obtaining the knockout plasmid pWJW202

[0084] Using the P.putidaKT2442 genome as a template, the upstream and downstream fragments of the PP3126-PP3142 gene cluster were amplified with corresponding primers. SacI and XbaI were introduced at the 5' and 3' ends of the upstream fragment, respectively, and BamHI and SacI restriction sites were introduced at the 5' and 3' ends of the downstream fragment, respectively. Using pDTW202 as a template, with primers

[0085] kan-lox-XbaI(+)acctctagaAATACGACTCACTATAGGGCG,

[0086] Kan-lox-BamHI(-)cgggatccGCGCAATTAACCCTCACTAAAG,

[0087] The kan cassette containing loxL / R sites at both ends was amplified, and XbaI and BamHI restriction sites were introduced at the 5' and 3' ends, respectively. The upstream fragment of the PP3126-PP3142 gene cluster was purified by SacI and XbaI digestion, the downstream fragment was purified by BamHI...

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Abstract

The invention discloses a pseudomonas putida gene knockout and genome simplification system, and belongs to the field of microbial gene engineering. According to the pseudomonas putida gene knockout and genome simplification system disclosed by the invention, a suicide plasmid knockout system and a site specific recombination system are skillfully combined, so that the correction rate of two turns of screening is greatly improved to be higher than 90%, and the correction rate is still higher than 90% when a gene cluster, which is 69 genes long, is knocked out. The system disclosed by the invention achieves the continuous knockout of the gene cluster, simple and convenient to operate, efficient, high in correction rate and low in cost. The system disclosed by the invention lays a molecular basis for the simplification and the optimization of a pseudomonas putida genome; therefore, the system is conducive to the implementation of efficient construction of chassis cells of synthetic biology.

Description

technical field [0001] The invention relates to a Pseudomonas putida gene knockout and genome simplification system, in particular to a knockout system based on the combination of homologous recombination and specific site recombination, belonging to the field of microbial genetic engineering. Background technique [0002] Pseudomonas putida is a Gram-negative bacterium that is mainly used in the production of polyhydroxyalkanoate (PHA), which is widely used in the production of bioplastics and high value-added medical materials. In order to improve the production characteristics of the strain itself, in addition to conventional metabolic engineering, it is imperative to optimize its genome. From the perspective of synthetic biology, streamlining and optimizing the genome of this strain is an important means to optimize it as a production chassis cell. Therefore, it is particularly important to construct an efficient, fast and accurate gene cluster continuous knockout system...

Claims

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Application Information

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IPC IPC(8): C12N15/78C12N15/66C12N1/21C12R1/40
CPCC07K14/21C12N15/66C12N15/78
Inventor 王小元王建莉马文渐林琳王雨舟王甜忆黄舒婷王雨倩张怡平孙康康李烨
Owner JIANGNAN UNIV
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