Method for randomly knocking out streptomyces genome DNA large fragment in vivo

A technology of large fragments and genomes, applied in the field of genetic engineering, can solve problems such as knockouts that cannot be knocked out with large fragments of genomes

Inactive Publication Date: 2014-10-22
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The large segment knockout system of Streptomyces can only target and knock out specific segments on the chromosome; although the miniature transposon system is efficient and convenient, it cannot be used for genome large segment knockout

Method used

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  • Method for randomly knocking out streptomyces genome DNA large fragment in vivo
  • Method for randomly knocking out streptomyces genome DNA large fragment in vivo
  • Method for randomly knocking out streptomyces genome DNA large fragment in vivo

Examples

Experimental program
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Effect test

Embodiment 1

[0037] A method for randomly knocking out large fragments of Streptomyces genomic DNA in vivo, the flow chart is as follows figure 1 shown, including the following steps:

[0038] 1) Plasmid construction: Recombinant plasmids such as pTNL_101, pTNL_102, pTNL_103, pTNL_104 and pTNL_105 were completed on the basis of pDZY101 (CN102154268A) by PCR and conventional molecular cloning methods.

[0039] pTNL_101: Using pDZY101 as a template, PCR fragments with integrase, ermE promoter, IRR, oriT, apramycin resistance gene fragments and oriC were cloned into sceS The incomplete kanamycin resistance gene fragment at the restriction site (the name is aphII-S, the sequence is shown in SEQ ID NO.1), and its structure is shown in figure 2 shown.

[0040] The construction methods of pTNL_103, pTNL_104 and pTNL_105 are the same as pTNL_101, the difference is that the promoters that start the transposase are different, which are neo promoter, hrdB Gene promoter and tcp830 inducible pro...

Embodiment 2

[0048] Transform the plasmids pTNL101 and pDZY101 into Escherichia coli ET12567, pick a single colony and insert them into 5mL LB liquid medium for overnight culture, transfer 1% of the inoculum to 100mL LB medium, and cultivate to OD at 37°C and 200rpm 600The value is about 0.3. After centrifugation, use 2×YT medium to wash twice, add 3mL 2×YT medium to mix the cells, and divide into 6 equal parts for later use. Use 6 mL of 2×YT medium, add 120 μL of Streptomyces coelicolor spore suspension, treat at 50°C for 10 minutes, divide into 6 equal parts, mix with the above bacteria, and then centrifuge to coat MS solid medium. After culturing at 30°C for 20 h, spread nalidixic acid and apramectin antibiotics on three pTNL101-transferred Streptomyces coelicolor M145 solid MS medium (final concentrations were 30 μg / mL and 50 μg / mL, respectively), 3 Nalidixic acid and apramectin (final concentrations of 30 μg / mL and 50 μg / mL, respectively) were coated on the solid MS medium of Streptom...

Embodiment 3

[0053] Transform the plasmids pTNL103 and pDZY101 into Escherichia coli ET12567, pick a single colony and insert them into 5mL LB liquid medium for overnight culture, transfer 1% of the inoculum to 100mL LB medium, and cultivate to OD at 37°C and 200rpm 600 The value is about 0.3. After centrifugation, use 2×YT medium to wash twice, add 3mL 2×YT medium to mix the cells, and divide into 6 equal parts for later use. Use 6 mL of 2×YT medium, add 120 μL of Streptomyces coelicolor spore suspension, treat at 50°C for 10 minutes, divide into 6 equal parts, mix with the above bacteria, and then centrifuge to coat MS solid medium. After culturing at 30°C for 20 h, spread nalidixic acid and apramectin antibiotics on three pTNL101-transferred Streptomyces coelicolor M145 solid MS medium (final concentrations were 30 μg / mL and 50 μg / mL, respectively), 3 Nalidixic acid and apramectin (final concentrations of 30 μg / mL and 50 μg / mL, respectively) were coated on the solid MS medium of Strepto...

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Abstract

The invention discloses a method for randomly knocking out a streptomyces genome DNA large fragment in vivo. The method comprises the following steps: (1) constructing series of recombinant plasmids pTNL_101, pTNL_103, pTNL_104 and pTNL_105 as a first sceS enzyme cutting site random introduced plasmid; (2) introducing the constructed recombinant plasmids pTNL_101, pTNL_103, pTNL_104 and pTNL_105 into streptomyces coelicolor M145, and constructing a mutant sub-library 1 without random chromosome large fragment; (3) constructing the series of recombinant plasmids pTNL_102 as a second sceS enzyme cutting site random introduced plasmid, and introducing into the mutant sub-library 1 to obtain a mutant sub-library 2; (4) screening out large fragment knockout muton by using an R2YE flat panel, precisely positioning a chromosome deletion region segment, and performing positioning knockout in an original strain to perform verification. The method disclosed by the invention can be used for removing removable parts of a chromosome region effectively and randomly; at present, a positioning result shows that the lengths of the knockout fragments are between 2kb and 100kb, thus an evidence for transforming streptomyces into antibiotic heterologous expression hosts and chassis organisms can be provided.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for randomly knocking out large fragments of Streptomyces genome DNA in vivo. Background technique [0002] Streptomyces are Gram-positive bacteria with a high GC content and are a large group of actinomycetes. Streptomyces have a complex cycle of development and differentiation, growing filamentously and producing spores on solid media. About 6,000 kinds of antibiotics and physiologically active substances have been identified in different species of Streptomyces, accounting for about half of the reported microbial-derived drugs (Berdy J. 2005. Bioactive microbial metabolites. J Antibiot (Tokyo) 58(1): 1- 26). Streptomyces coelicolor is the model strain of Streptomyces research, and the genetic background research is relatively clear. In 2002, the entire genome of Streptomyces coelicolor was sequenced (Bentley S. D, K. F. Chater, A. M. Cerdeno-Tarraga, G....

Claims

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Application Information

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IPC IPC(8): C12N15/76
Inventor 路志群邢述永李文雪邓自发
Owner NANTONG UNIVERSITY
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