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Plasmid T5 having ampicillin resistance, DNA sequence, and construction method thereof

A technology with ampicillin and ampicillin, which is applied in the directions of recombinant DNA technology, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems such as the discovery and construction of ampicillin resistance genes that have not yet been reported.

Inactive Publication Date: 2011-04-13
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the ampicillin resistance genes discovered and applied at the present stage come from land (7, Feng Youjun, Zhang Huimin, Genetic Analysis and Identification of Multiple Resistance to Antibiotics Endowed by a Wild Plasmid. Journal of Shaanxi University of Science and Technology, 2003.21: 54-58) , but the discovery and construction of the ampicillin resistance gene and its plasmid from the deep sea have not been reported so far

Method used

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Examples

Experimental program
Comparison scheme
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Embodiment Construction

[0068] 1) Use CaCl 2Methods Prepare E.coli DH5α competent cells; E.coli DH5α can also be any E.coli genetically engineered bacteria, such as E.coli M15, E.coli TOP10F', E.coli BL21(DE3)plysS, E.coli BL21(DE3)plysS, E.coli coli JM109, etc.;

[0069] 2) Mix 1 ng of plasmid T5 and 200 μl of competent cells evenly, after 30 min in ice bath, heat shock at 42°C for 90 s, take it out immediately and place in ice bath; All operations (including steps 2-6) are consistent with the experimental group;

[0070] 3) Add 800 μl of LB liquid medium, mix gently, and shake and culture at 37°C, 100 rpm for 1 hour; the composition of LB liquid medium is: 1% NaCl, 1% Tryptone Powder, 0.5% Yeast Extract;

[0071] 4) Centrifuge at 2000rpm for 2.5min, and absorb 800μl of supernatant in an ultra-clean bench;

[0072] 5) Gently mix the transformation product with a pipette with the tip cut off, draw the transformation product and place it on the LB liquid medium (containing 100 μg / ml ampicillin) pla...

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Abstract

Plasmid T5 having ampicillin resistance, DNA sequence, and construction method thereof are disclosed in the invention. The plasmid derived from marine bottom sediment is transformed into a colibacillus competent cell, so that the cell has the characteristic of ampicillin resistance; the plasmid T5 can be stably bred in various E.coli cells and can be obtained by conventional plasmid extracting technology. The construction method comprising the steps of: extracting marine bottom sediment sample metagenome DNA, incomplete digesting, testing and recovering the 1-5kb segment; self connecting the digested DNA segment; transforming the connected DNA segment into the competent cell, screening and selecting the grown colony, extracting and verifying the plasmid; adopting the plasmid as the template, performing PCR amplification by adding a primer to obtain a subsequence of the plasmid; and finally obtaining the complete sequence by reverse sequencing.

Description

technical field [0001] The invention relates to a plasmid T5 with ampicillin resistance, in particular to a plasmid T5 and DNA sequence derived from deep sea sediment metagenomic DNA and a construction method thereof. Background technique [0002] Methods for screening functional genes from samples usually include methods based on microbial isolation and culture technology and metagenomic technology. Existing studies believe that only less than 1% of microorganisms in nature can be isolated and cultured with existing technical means (1, Berthelet, M., L.G Whyte, and C.W.Greer, Rapid, direct extraction of DNA from soils for PCR Analysis using polyvinylpolypyrrolidone spin columns.FEMS Microbiol Lett, 1996.138(1):17-22; 2, Coolen, M.J.and J.Overmann, Analysis of subfossil molecular remains of purple sulfur bacteria in a lake sediment.Appl Environ Microbiol, 1918.64(1 4513-4521), while the sample metagenomic DNA contains culturable and non-culturable microbial genes, expanding...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/64C12N15/70C12R1/19
Inventor 曾润颖陈兴麟
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION