Establishment method of streptomyces diastatochromogenes expression system

A Streptomyces chromogenes and expression system technology, applied in the field of genetic engineering, can solve the problems of low transcriptional activity, ineffectiveness, and lack of versatility of promoters

Inactive Publication Date: 2013-09-11
CHINA JILIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the metabolic process of Streptomyces is a very complex, inter-coupled, multi-level regulatory process. Therefore, the promoters of Streptomyces genes generally have complex transcription patterns. Therefore, PermE* Promoters are not universal in Streptomyces, some studies reported PermE* Promoter transcriptional activity is low or even ineffective
Especially for amylase Streptomyces chromogenes, there is no effective expression system

Method used

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  • Establishment method of streptomyces diastatochromogenes expression system
  • Establishment method of streptomyces diastatochromogenes expression system
  • Establishment method of streptomyces diastatochromogenes expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Using the green fluorescent protein gene gfp Construction of the genetic expression system of amylase Streptomyces chromogenes as a reporter gene:

[0036] Using plasmid pCAMBIA1302 as template, P gfp f Eco R I and P gfp R Hin dIII is the primer PCR obtained containing Eco R I and Hin dIII two enzyme cutting sites gfp Fragment, connected with pMD18-T Vector to construct cloning vector pMD18-T- gfp ( Eco R I+ Hin dIII). The cloning vector pMD18-T- gfp ( Eco R I+ Hin dIII) converted to E. coli JM109 recipient bacteria were spread on the LB agar plate containing Amp, IPTG, and X-gal. After culturing overnight at 37 °C, many white colonies grew on the plate. Randomly pick white clones for enzyme digestion identification and send them to Shanghai Sangon Bioengineering Co., Ltd. for sequencing Eco R I and HindIII double digestion to obtain Eco R I and Hind III of the two enzyme cleavage sites gfp The fragment was ligated wit...

Embodiment 2

[0042] Example 2: Using the green fluorescent protein gene gfp Preliminary evaluation of the genetic expression system of amylase Streptomyces chromogenes as a reporter gene:

[0043] Pick the colonies of Streptomyces amylase chromogenes grown on the plate containing 50 μg / mL apramycin to inoculate the CP medium, shake overnight at 28 °C and 180 r / min, and then inoculate them in the Fill with 50 mL of fresh CP medium, culture to mid-logarithmic growth, dry cell weight (DCW) is about 0.3 g / L, collect mycelium by centrifugation at 6000 r / min, wash mycelium twice with PBS buffer Afterwards, resuspend in 30 mL PBS. Take 5-10 μL bacterial liquid smear, observe under Olympus BX50 fluorescence microscope, and take pictures with Color Video Camera of SONY 3CCD at the same time. Using wild bacteria as a control, select the excitation filter to be 485 nm and the emission filter to be 520 nm to determine the transcriptional activity of the promoter.

[0044] The results showed that t...

Embodiment 3

[0045] Example 3: Vitella hyaline hemoglobin gene vgb Expression in amylase Streptomyces chromogenes:

[0046] recombinant plasmid pIB139- vgb builds like Figure 6 As shown, the steps are related to the recombinant plasmid pIB139- vgb The method of construction is similar.

[0047] like Figure 7 and Figure 8 Shown, respectively, recombinant plasmid pET28a- vgb and pIB139- vgb Enzyme digestion verification results. Recombinant plasmid pET28a- vgb through Eco R I and Hin DNA fragments of about 0.5kb and 5.3kb were obtained after dIII double digestion, respectively vgb The size of the gene fragment is the same as that of the plasmid pET28a. recombinant plasmid pIB139- vgb through Nde I and not I obtained a DNA fragment of about 0.6kb after double enzyme digestion, and vgb Gene fragments are of the same size. The above results prove that the recombinant plasmid pIB139- vgb builds correctly.

[0048] Extract plasmid pIB139- vgb , into amylase S...

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Abstract

The invention discloses an establishment method of a streptomyces diastatochromogenes expression system. The establishment method comprises the following steps of: screening a necessary or efficient ingredient of the streptomyces diastatochromogenes expression system by virtue of a green fluorescent protein gene gfp at first, and then constructing the expression system of a target gene, wherein the necessary or efficient ingredient is erythromycin resistance genome constructive promoter permE*; the target gene promotes production of toyocamycin; and the target gene is a haemoglobin gene vgb. The construction processes are as follows: 1) constructing a carrier pIB139-gfp; and 2) integrating the expression carrier into a streptomyces diastatochromogenes chromosome by virtue of a conjugational transfer method, so as to obtain a recombinant bacterium. The invention provides an efficient screening method and application of the streptomyces diastatochromogenes expression system. Compared with the original strain, the recombinant bacterium has the advantages that the toyocamycin yield of the recombinant bacterium is increased by at least 21.2%, and the concentration of the recombinant bacterium is increased by 11.6%.

Description

technical field [0001] The invention relates to a method for establishing an expression system of amylase streptomyces chromogenes, and belongs to the technical field of genetic engineering. Background technique [0002] Toyocamycin is a new type of nucleoside antibiotics with the molecular formula C 12 h 13 N 5 o 4 , Ribose C 1 The deazapurine ring similar to guanine is connected, and the core structure is a pyrropyrimidine nucleoside analogue. The mechanism of action is mainly to affect the growth of bacteria by inhibiting the transcription of microorganisms, and the research reports on its biological activity are mainly concentrated in the field of clinical medicine. Recent studies have found that toyocamycin has a good control effect on a variety of plant diseases, long-term use will not cause environmental pollution, and it also has a certain regulatory effect on plant growth. Therefore, toyocamycin has great application potential in the field of agricultural plan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/76C12R1/525
Inventor 马正俞晓平刘金秀申屠旭萍边亚琳郝培应许益鹏
Owner CHINA JILIANG UNIV
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