Method for preparing radio-resistant bacterium having luciferase activity

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...

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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

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