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Method for preparing radio-resistant bacterium having luciferase activity

A technology of anti-radiation bacteria and luciferase, applied in the biological field, can solve the problems of unavoidable pollution of the environment, inability to exist stably, and a small amount of protein, and achieve the effect of wide application prospects.

Inactive Publication Date: 2008-01-30
ZHEJIANG UNIV
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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 bacterium Deinococcus radiodurans-Escherichia coli shuttle vector pRAD1 (developed with the replicon of the plasmid pUE10 derived from D. radiodurans Sark strain and the Escherichia 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 radioresistant bacteria Deinococcus radiodurans with pRAD1; at the same time, pRAD1 can be stable in the selective medium containing the antibiotic chloramphenicol Replication cannot exist stably in non-selective medium without chloramphenicol. This shortcoming is particularly problematic when releasing the radioresistant bacteria with foreign genes into the open environment in the field: soil and wastewater contaminated by radioactive substances, etc. It is very difficult to maintain a certain concentration of antibiotics in the environment, and it is very difficult to put antibiotics in the field, and it is very likely that there will be no necessary antibiotic-resistant bacteria, and at the same time cause secondary pollution of the environment; secondly, although as a substitute for the open environment in the field, A large-scale closed environment can be used to deal with pollutants, but the use of a large amount of antibiotics will inevitably pollute the environment

Method used

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  • Method for preparing radio-resistant bacterium having luciferase activity
  • Method for preparing radio-resistant bacterium having luciferase activity
  • Method for preparing radio-resistant bacterium having luciferase activity

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Preparation of plasmids

[0029] (1) Isolation and purification of plasmid

[0030] 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. The plasmid components were extracted with the QIAfilter Plasmid kit (QIAGEN, Germany), and separated and purified by agarose gel electrophoresis. As a result, a plasmid of about 2.45 kb was found. Mackay et al. (Arch. Microbiol., 141:91-94, 1985) observed the radioresistant 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.

[0031] (2) Determination of base sequence

[0032] 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 pUE3...

Embodiment 2

[0033] Embodiment 2: Construction of shuttle vector pZT15, pZT17

[0034] (1) Construction of pZT15

[0035] 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 to 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.

[003...

Embodiment 3

[0038] Example 3: Verification of the properties of the shuttle vectors pZT15 and pZT17 of the present invention

[0039] (1) Recombinant transformation of the radioresistant strain Deinococcus grandis ATCC43672 using a shuttle vector

[0040] The above-mentioned shuttle vector pZT15 or pZT17 was used to recombinantly transform the radioresistant strain Deinococcus grandis ATCC43672, and the recombinant transformation referred to the method of Kitayama et al. (J. Bacteriol., 155: 1200-1207, 1983), using the CaCl2 method. 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.

[0041] (2) Stability verification of the shuttle carrier in the radioresistant bacteria Deinococcus grandis ...

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Abstract

The invention relates to a preparation method of radioresistant bacterium that contains the activity of luciferase. The invention takes plasmids originating from radioresistant bacterium deinococcus radiopugnans as a base to recompose with plasmids that can ensure self-duplication in escherichia coli to form a shuttle carrier. The carrier is recomposed with DNA fragments in the luciferase gene lux field that originates from into host DNA of photinus pyralis from North America into plasmids with luciferase genes. Then the plasmids are introduced into bacterial of a radioresistant bacterium family, deinococcus grandis are transformed by the constructed plasmids to get chloramphenicol resistant strains. The radioresistant bacterium prepared by the method of the invention can be used in various fields of outdoor opening and close environments. The carrier has the luciferase genes, thereby having better application value to real-time supervision and control on the propagation, distribution and influence to the environment of the recomposed transformants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing a gene carrier and a transformant thereof, that is, a method for preparing a radiation-resistant bacterium with luciferase activity. 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 that of E. coli an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/53
Inventor 屠振力
Owner ZHEJIANG UNIV
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