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Plasmid with bar streptomycete Lat gene loss, derivative and constructing method thereof

A technique for gene deletion of Streptomyces clavicularis, applied in the field of bioengineering, can solve the problems of no single crossover integration into chromosome, lat gene deletion mutation, cumbersome steps, etc., and achieve the construction method is simple and easy, with strong purpose and high work intensity small effect

Inactive Publication Date: 2005-07-27
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the relatively cumbersome steps of the insertion mutation method used, and the shuttle plasmid vector pIJ486 used, it does not have the ability to integrate into the chromosome by single crossover, while double crossover integration is bound to be difficult.
[0006] However, there is no report on the deletion mutation of the lat gene of Streptomyces clavulicularis and the selection of a shuttle plasmid vector with single crossover integration ability to construct a lat gene mutant plasmid

Method used

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  • Plasmid with bar streptomycete Lat gene loss, derivative and constructing method thereof
  • Plasmid with bar streptomycete Lat gene loss, derivative and constructing method thereof
  • Plasmid with bar streptomycete Lat gene loss, derivative and constructing method thereof

Examples

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Embodiment 1

[0036] Construction of recombinant plasmid pKCLHS

[0037] ① Extraction of total DNA of Streptomyces clavulatus

[0038] Pick the solid ISP (ISP (Difco): 0.8%, agar powder: 2%, pH = 7.3) plate Streptomyces clavulatus cultured at 28°C for 3 days and inoculate it in the liquid ISP medium, culture it on a shaker at 28°C for 2 days, centrifuge The bacterial cells were harvested, and the bacterial cells were washed once with 10 mM EDTA at pH=8.0 or twice with water. Add SET (10mmol / L Tris-HCl (pH8.0), 10mmol / L NaCl, 1mmol / L EDTA (pH8.0)) solution to 1mL, and add 20μL of 50mg / mL (or an equivalent amount of other concentrations of lysozyme ), 37°C water bath for 30-60min. Add 28 μL of 20 mg / mL proteinase K and mix well, add 120 μL 10% SDS, invert and mix well, and incubate at 55° C. for 2 hours. Divide the mixture into two clean centrifuge tubes (0.8mL / tube), add 500μL of phenol / chloroform respectively, shake vigorously and mix thoroughly, and carefully draw the supernatant into a...

Embodiment 2

[0055] Construction of recombinant plasmid pKCLES

[0056] For the specific method, see ①, ②, ③, and ④ four steps in Example 1, and the ⑤ step is different from the restriction endonuclease used in the ⑤ step in Example 1, as follows:

[0057] The resulting recombinant plasmid pKC1139-lat was subjected to EcoRI-ScaI double enzyme digestion, a section of EcoRI-ScaI gene fragment with a size of about 1.4kb was cut out of the lat gene, and about 7.0kb linear pKCLES fragment was reclaimed (DV805A glue recovery kit of Takara Company), Use T for both cohesive ends 4 DNA polymerase (product of Takara company) fill level, fill 805A gel recovery kit), use T for its two sticky ends 4 DNA polymerase (product of Takara Company) was used to make up the level, and the leveling system was as follows: sterilized double-distilled water: 3.5 μL, T4 DNA polymerase buffer (10×): 1 μL, 0.1% BSA: 1 μL, recovered pKCLES: 2.5 μL, dNTP (2mM): 1μL, the total system of filling up reaction: 9μL; the ...

Embodiment 3

[0061] Derivative 1 of Escherichia coli-Streptomyces shuttle plasmid containing partial deletion of Streptomyces clavulicularis lat gene and its construction method

[0062] For the specific method, see ①, ②, ③, and ④ four steps in Example 1, the ⑤ step is different from the restriction endonuclease used in the ⑤ step in Example 1, and the specific steps are as follows:

[0063] Adopt the rest of the enzyme cutting sites on the lat gene except HindIII-ScaI and EcoRI-ScaI used for constructing plasmid pKCLHS and pKCLES (see image 3 Any two enzymes in ), such as using EcoNI-HindIII to cut the lat gene in the plasmid pKC1139-lat, cut off a EcoNI-HindIII gene fragment with a size of about 0.7kb of the lat gene, and reclaim about a 7.6kb linear fragment, The two sticky ends were filled with T4 DNA polymerase (product of Takara Company), and the filling system was as follows: sterilized double-distilled water: 3.5 μL, T4 DNA polymerase buffer (10×): 1 μL, 0.1% BSA: 1 μL , DNA frag...

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Abstract

Plasmid with lat gene deletion of claviform streptomyces, its derivative and constructing method are disclosed. Upstream and downstream inducers are designed to in vitro amplify lat gene, constructing plasmid and derivative with single exchage and assembly, deletion mutating lat gene with influence on clavulanic acid yield, and constructing in colibacellus. The said plasmid contains a part of encoded lysine e-transaminase gene and duplication inducing points of plasmids of colibacillus and streptomyces, resistant selective marks of ambylan expressed in colibacillus and streptomyces, and moderate sensitive duplication sub-gene.

Description

technical field [0001] The invention belongs to bioengineering, and relates to a plasmid, a derivative and a construction method for realizing PCR in vitro amplification primers for encoding lysine epsilon aminotransferase lat gene of Streptomyces clavulatus and deletion of the lat gene by using genetic engineering technology. Background technique [0002] Since the advent of penicillin, β-lactam antibiotics have been widely used so far, and many β-lactam varieties with new characteristics have come out continuously. However, with the widespread use of β-lactam antibiotics, the resistance of various bacteria to this class of antibiotics is also increasing. There are many bacterial resistance mechanisms, among which the production of β-lactamase is the most important mechanism of drug resistance, so the development of β-lactamase inhibitors is of great significance for solving the problem of bacterial drug resistance. This kind of enzyme inhibitor is used in combination with...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/74
Inventor 王艳萍左志晗金守光
Owner TIANJIN UNIV OF SCI & TECH
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