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A kind of conjugative transfer method of Saccharopolyspora rubrum industrial production strain

A technique of conjugative transfer of Saccharopolyspora rubrum, which is applied in the field of microorganisms, can solve the problems of little known, unknown gene function of Saccharopolyspora rubrum, and unreported efficiency of conjugative transfer, so as to improve transfer rate and improve conjugative transfer efficiency effect

Active Publication Date: 2020-03-24
HEC PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 2007, Markiyan Oliynyk published the whole genome sequencing information of the type strain NRRL23338 of Saccharopolyspora rubrum in "Nature", which created conditions for the study of gene function of Saccharopolyspora rubrum, but so far there are still a large number of Saccharopolyspora rubrum The function of the gene is unknown, especially the regulatory genes related to erythromycin synthesis are poorly understood
[0005] At present, there are many reports on the establishment of a genetic operating system for S. What is more important is the conventional conjugative transfer condition in Streptomyces (Bierman M, et al.Gene, 1992,116 (1): 43-49), although report this conjugative transfer method can be successful, but conjugative transfer efficiency is very low, is not suitable for The genetic operating system used to establish Saccharopolyspora rubrum has certain limitations for studying gene functions in vivo; Yun Chen disclosed a method for conjugative transfer using a temperature-sensitive shuttle plasmid (Chen, Yun, et al.Applied and environmental microbiology, 2008,74(6):1820-1828), and applied it to the genetic modification of Saccharopolyspora red strain, but its conjugative transfer efficiency has not been reported, and it is limited to the use of temperature-sensitive shuttle vectors, for Other vectors, such as integration-type and suicide-type shuttle plasmids, are not applicable, which limits the application of this method to a certain extent

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Escherichia coli S17-1 (containing the pSET152 shuttle plasmid) was used as the donor bacteria, cultured overnight to OD 600 0.4 to 0.6, centrifuge to collect 25 mL of bacteria, rinse the bacteria twice with an equal volume of sterile LB, and suspend in 0.1 times the volume of LB for later use. Take about 1×10 8 The spores of an industrial production strain of Saccharopolyspora rubrum were washed twice with LB, suspended in 500 μL TES Buffer, heat-shocked at 50°C for 10 min, and then added an equal volume of optimized TSB liquid medium (Mg 2+ The concentration is 8mM), germinated in a static water bath at 37°C for 3h. Wash twice with LB, and finally suspend in 1mL LB as recipient bacteria. According to the ratio of the number of donor bacteria and recipient bacteria cells is about 3:1, take an appropriate amount and mix it evenly, spread 2CMY solid plate medium, cultivate overnight at 30°C for 22h, and then use nalidixic acid (final concentration: 50μg / mL) and apramy...

Embodiment 2

[0035] Escherichia coli S17-1 (containing the pSET152 shuttle plasmid) was used as the donor bacteria, cultured overnight to OD 600 0.4 to 0.6, centrifuge to collect 25 mL of bacteria, rinse the bacteria twice with an equal volume of sterile LB, and suspend in 0.1 times the volume of LB for later use. Take about 1×10 8 The spores of an industrial production strain of Saccharopolyspora rubrum were washed twice with LB, suspended in 500 μL TES Buffer, heat-shocked at 50°C for 10 min, and then added an equal volume of optimized TSB liquid medium (Mg 2+ The concentration is 12mM), and germinated in a static water bath at 37°C for 6h respectively. Wash twice with LB, and finally suspend in 1mL LB as recipient bacteria. According to the ratio of the number of donor bacteria and recipient bacteria cells is about 7:1, take an appropriate amount and mix it evenly, spread 2CMY solid plate medium, cultivate overnight at 34°C for 22h, and then use nalidixic acid (final concentration: 50μg...

Embodiment 3

[0037] Escherichia coli S17-1 (containing the pSET152 shuttle plasmid) was used as the donor bacteria, cultured overnight to OD 600 0.4 to 0.6, centrifuge to collect 25 mL of bacteria, rinse the bacteria twice with an equal volume of sterile LB, and suspend in 0.1 times the volume of LB for later use. Take about 1×10 8 The spores of an industrial production strain of Saccharopolyspora rubrum were washed twice with LB, suspended in 500 μL TES Buffer, heat-shocked at 50°C for 10 min, and then added an equal volume of optimized TSB liquid medium (Mg 2+ The concentration is 10mM), and cultivated in a static water bath at 37°C for 3h. Wash twice with LB, and finally suspend in 1mL LB as recipient bacteria. According to the ratio of the number of donor bacteria and recipient bacteria cells is about 5:1, take an appropriate amount to mix, spread 2CMY solid plate medium, and culture overnight at 32°C for 24h, then use nalidixic acid (final concentration: 50μg / mL) and apramycin (50 ...

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Abstract

The invention discloses a method for conjugational transfer of a saccharopolyspora erythraea industrial producing strain. The method comprises the steps: selecting mature saccharopolyspora erythraea spores, pretreating, then putting in a TSB liquid culture medium containing Mg<2+>, germinating in static water bath, then cleaning, and carrying out suspension treatment to obtain receptor bacteria; and mixing donor bacteria and receptor bacteria spores, culturing in a coated 2CMY solid conjugational transfer plate culture medium, and growing to obtain a conjugational transfer factor. The method has the advantages of convenience in operation, and simple and easy implementation, and the conjugational transfer efficiency of saccharopolyspora erythraea can reach 2*10<-5>, the situation of difficult genetic manipulation of the saccharopolyspora erythraea industrial producing strain can be well overcome, and convenient conditions are provided for directional genetic modification and gene function research of the saccharopolysporaerythraea industrial strain.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a method for conjugative transfer of Saccharopolyspora rubrum industrial production strains. Background technique [0002] Red Saccharopolyspora (Saccharopolyspora erythraea) was originally isolated from soil by McGuire et al. in 1952. It is a Gram-positive actinomycete belonging to the genus Saccharopolyspora and has a high biological homology. In recent years, it has received extensive attention and intensive research because of its use in the production of erythromycin on an industrial scale. In 2007, Markiyan Oliynyk published the whole genome sequencing information of the type strain NRRL23338 of Saccharopolyspora rubrum in "Nature", which created conditions for the study of gene function of Saccharopolyspora rubrum, but so far there are still a large number of Saccharopolyspora rubrum Gene function is unknown, especially the regulatory genes related to erythromycin synthesis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/03C12R1/01C12R1/19
CPCC12N15/03
Inventor 赵裕栋鲍素敏
Owner HEC PHARM CO LTD
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