Shuttle vector between antiradiation bacterium and colibacillus and its construction process

A technology of Escherichia coli and anti-radiation bacteria, applied in the biological field, can solve the problems of inevitable pollution of the environment, difficulty in resisting biological substances, and inability to control copy number, etc., and achieve the effect of wide application prospects

Inactive Publication Date: 2007-06-27
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there has been no research on the transformation of bacteria of the genus Deinococcus other than Deinococcus radiodurans (such as the above-mentioned D.radiopugnans; D.grandis, etc.) with plasmid vectors containing foreign genes.
In addition, there are some disadvantages in the production of the above-mentioned recombinant transformants: the use of an integrated plasmid that cannot replicate itself in Deinococcus bacteria, the copy number cannot be controlled, the amount of expressed protein is small, and antibiotics need to be added to the culture medium, etc.
In order to overcome these shortcomings, the radioresistant bacteria Deinococcus radiodurans-Escherichia coli shuttle vector pRAD1 (developed with the replicon of the plasmid pUE10 derived from the D. radiodurans Sark strain and the E. coli vector pMTL23) (Meima & Lidstrom, Appl.Environ. Microbiol., 66:3856-3867, 2000), but there is no research report on the recombinant transformation of Deinococcus bacteria other than the rad

Method used

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  • Shuttle vector between antiradiation bacterium and colibacillus and its construction process
  • Shuttle vector between antiradiation bacterium and colibacillus and its construction process
  • Shuttle vector between antiradiation bacterium and colibacillus and its construction process

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Embodiment 1

[0029] Example 1: Preparation of plasmids

[0030] (1) Isolation and purification of plasmid

[0031] The radioresistant bacterium Deinococcus radiopugnans ATCC19172 was cultured with TGY medium (containing 0.5% tryptone, 0.1% glucose, and 0.3% yeast extract), and the cells were collected by centrifugation. Plasmid components were extracted with QIAfilter Plasmid kit (QIAGEN, Germany), separated and purified by agarose gel electrophoresis, and a plasmid of about 2.45 kb was found. Mackay et al. (Arch. Microbiol., 141:91-94, 1985) observed the radiation-resistant bacteria Deinococcus radiopugnans with an electron microscope and reported the presence of a plasmid pUE30 with a size of about 2.5 kb. Therefore, the purified plasmid of 2.45 kb was isolated as pUE30 this time.

[0032] (2) Determination of base sequence

[0033] The restriction map of the refined pUE30 above was made, and it was found that there was a restriction site for HincII and Aor51HI in pUE30 respectively. ...

Embodiment 2

[0034] Embodiment 2: Construction of shuttle vector pZT15, pZT17

[0035] (1) Construction of pZT15

[0036] Using pUE30 as a template, DNA synthesized from the base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 (designed by yourself based on the base sequence of pUE30 above) as primers, and using AmpliTaq Gold DNA polymerase (AppliedBiosystems) for PCR reaction , to obtain a PCR product with a restriction endonuclease SphI site designed at both ends and a complete base sequence of pUE30. Next, the plasmid pKatCAT (Funayama et al., Mutat.Res ., 435:151-161, 1999), and then mixed with the SphI digest of the above PCR product, and ligated with DNA ligase. The above linker was transformed into Escherichia coli JM109 strain by electroporation. A strain with a plasmid (about 6.1 kb in size) connected with pUE30 and pKatCAT was selected from the Amp-resistant transformant, and the plasmid was named as the shuttle vector pZT15. The structure of pZT15 is shown in Figure 1.

[0...

Embodiment 3

[0039] Example 3: Verification of the properties of the shuttle vectors pZT15 and pZT17 of the present invention

[0040] (1) Recombinant transformation of the radioresistant strain Deinococcus grandis ATCC43672 using a shuttle vector

[0041] Use the above-mentioned shuttle vector pZT15 or pZT17 to recombine transform the radioresistant bacteria Deinococcus grandis ATCC43672 strain, and refer to the method of Kitayama et al. 2 Law. No matter using pZT15 or pZT17 shuttle vector, strains containing chloramphenicol resistance gene can be obtained. Plasmid components were extracted from these strains using the QIAprep Miniprep Plasmid kit (Qiagen), and analyzed by agarose gel electrophoresis, and it was confirmed that the introduced plasmid remained in the recombinant transformant.

[0042] (2) Stability verification of the shuttle carrier in the radioresistant bacteria Deinococcus grandis

[0043] Cultivate the radioresistant bacterium Deinococcus grandis containing the shutt...

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Abstract

The present invention is shuttle vector between antiradiation bacterium and colibacillus and its preparation process. Antiradiation bacterium Deinococcus radiopugnans originated plasmid and the colibacillus plasmid capable of being auto replicated are recombined to constitute the shuttle vector. The shuttle vector is recombined with the DNA segment possessing luciferase gene lux originated from host DNA of Photinus pyralis to constitute plasmid with luciferase gene, the plasmid is introduced into antiradiation bacterium, and the constituted plasmid is used in converting Deinococcus grandis to obtain bacterium strain with chloromycetin resistance. The shuttle vector can auto replicate in antiradiation bacterium and colibacillus and may be introduced into antiradiation bacterium. It may be used in various kinds of field environments and possesses luciferase gene.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a gene carrier and a construction method thereof, that is, a shuttle carrier between the genus radioresistant bacteria and Escherichia coli and a construction method thereof. Background technique [0002] Different organisms have different resistance to radiation. There are microorganisms with strong resistance to radiation in nature. These microorganisms are usually called radiation-resistant bacteria, also known as radiation-resistant bacteria or radio-resistant Deinococcus. This article is hereinafter referred to as radiation-resistant bacteria. The representative radiation-resistant bacteria is the genus Deinococcus, and now there are 7 types of bacteria (D.radiodurans; D.radiopugnans; D.radiophilus; D.grandis; D.proteolyticus; D.geothermalis; D.murrayi) (Ferreira et al., Int. J. Syst. Bacteriol., 47:939-947, 1997). The resistance of these bacteria to radiation is about 100 times...

Claims

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Application Information

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IPC IPC(8): C12N15/70
Inventor 屠振力
Owner ZHEJIANG UNIV
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