Plasmid carrier construction method applied to Shewanella

A technology of Shewanella and plasmid vectors, which is applied in the field of construction of plasmid vectors for Shewanella, and can solve problems such as lack of and no major breakthroughs in Shewanella

Inactive Publication Date: 2016-04-20
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the genomics research and genetic engineering transformation of Shewanella have not made much breakthrough. One

Method used

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  • Plasmid carrier construction method applied to Shewanella
  • Plasmid carrier construction method applied to Shewanella
  • Plasmid carrier construction method applied to Shewanella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the cultivation of thalline

[0030] 1) Solid culture

[0031] The composition of LB medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L and agar 15g / L, streak inoculated on solid medium. Shewanella xiamenensis BC01 and Shewanellaoneidensis MR-1 were cultured at 30°C for 16h, and Escherichia coli DH5α were cultured at 37°C for 16h. If the bacterium carries the kanamycin resistance gene, the culture medium needs to add 1% kanamycin.

[0032] 2) Liquid culture

[0033] First, pick a single colony from the LB agar plate with an inoculation loop and inoculate it into a 250mL Erlenmeyer flask containing 50mL of LB culture solution. Shewanella xiamenensis BC01 and Shewanellaoneidensis MR-1 were cultured at 30°C for 16h, and Escherichia coli DH5α were cultured at 37°C for 16h. If the bacteria have a kanamycin resistance gene, the culture medium needs to be added with kanamycin, and the final concentration is 500mg / ml.

Embodiment 2

[0034] Embodiment 2: the extraction of plasmid

[0035] 1) Initial extraction of plasmids

[0036] Plasmid DNA was extracted from cultured Shewanella xiamenensis BC01 using OmegaPlasmidMiniKit kit. See the kit instructions for details.

[0037] 2) Agarose gel electrophoresis

[0038] Add 25ml of 1×TAEbuffer and 0.25g of agarose into the beaker. Heat it in a microwave oven to dissolve it completely, wait for it to cool to about 60°C, pour it into a cleaned glue-making tank, and insert a 50μL glue-making comb. After cooling and solidification, carefully pull out the comb. Put the gel together with the gel-making tank in the electrophoresis tank equipped with 1×TAEbuffer, with the side with the hole facing the negative electrode, and the liquid level of 1×TAEbuffer should completely submerge the gel. Take an appropriate amount of DNA sample, that is, mix the extracted pSXM33 and 6xloadingbuffer at a ratio of 5:1, add it to the sample well, and finally add 5-10 μL DNAmarker. ...

Embodiment 3

[0041] Example 3: Restriction Enzyme Digestion and Verification of Plasmids

[0042] In this experiment, fast cutting enzyme was used for enzyme digestion, and the enzyme digestion system was reacted in a water bath at 37° C. for 30 minutes, and then verified by agarose gel electrophoresis. The electrophoresis steps were the same as those described in Example 2.

[0043] The verification scheme is Lane2:XhoI, Lane3:HindIII, Lane4:PstI, Lane5:EcoRI, Lane6:EcoRV, Lane7:XhoI+EcoRI. After trying the above enzyme digestion method, the result is as follows figure 2 As shown, there are EcoRV, EcoRI and XhoI restriction enzyme sites in the pSXM33 plasmid sequence, and each site is unique on the plasmid.

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Abstract

The invention discloses a plasmid carrier construction method applied to Shewanella, and relates to Shewanella xiamenensis BC01 with a collection number of CGMCC No.11687. The method comprises the following steps: extracting a plasmid by the Shewanella xiamenensis BC01, purifying to obtain pSXM33, submitting the pSXM33 to inspection and sequencing; comparing obtained sequence information with NCBI database information, determining a distribution condition of an open reading frame on the pSXM33 plasmid, and drawing a map of the plasmid; intercepting a fragment with a duplication original part from the pSXM33 by a genetic engineering means, and connecting the intercepted fragment with a resistance and duplication origin fragment of a pET28a plasma to construct two plasmids, namely, pETSXM1 and pETSXM2. As proved, the pETSXM1 and the pETSXM2 can pass through escherichia coli and Shewanella MR-1.

Description

technical field [0001] The invention relates to Shewanella, in particular to a method for constructing a plasmid vector applied to Shewanella. technical background [0002] Plasmids are non-nuclear circular DNA molecules present in some bacteria or yeast, ranging in size from thousands to hundreds of thousands of bases, and usually contain an independent origin of replication, that is, capable of autonomous replication independent of nuclear chromosomes. Therefore, the plasmid can maintain a certain copy number in the progeny and express the gene it carries. Often plasmids carry genes that confer certain additional biochemical properties on the host, such as resistance to certain antibiotics. The above-mentioned characteristics make the plasmid can be used as a carrier in recombinant DNA technology, and the foreign gene recombined on the plasmid can be sent into the target host cell by genetic engineering means for reproduction and expression. Common artificial plasmid car...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/74C12N15/64C12R1/01
CPCC12N15/74C12N2800/101C12N1/205C12R2001/01
Inventor 周云丽吴意珣苏畅张霞卢英华
Owner XIAMEN UNIV
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