title: [Construction of double-labelled carbofuran-degrading bacterium Sphingomonas sp. CDS-1].
abstract: The genomic DNA of a carbofuran-degrading bacterium Sphingomonas sp. CDS-1 was digested by Sau3Al and ligated to pRobe-GFP digested by BamHI, and the product was transformed to the E. coli DH5alpha competent cells. Fifty positive clones that could emit green fluorescence under UV were selected from about 1 x 10(4) clones grown on selective plates AmpLB. One clone F7 with the strongest fluorescence was selected, the recombinant plasmid pF7 from this clone was digested with EcoR I & Hind III and the DNA fragment comprising gfp and promoter of Sphingomonas sp. CDS-1 was recovered, which was subsequently cloned into the broad host vector pPZP201 to construct a new plasmid pPZP201-gfp. pPZP201-gfp was introduced into Sphingomonas sp. strain CDS-1 by triparental conjugation to make strain CDS-gfp. gfp was expressed strongly and stably in strain CDS-gfp after 10 times successive re-culturing (48 h/time). The linA gene was inserted into Not I -cut transposon vector pUT/mini-Tn5 to construct a new transposon vector pUT/mini-Tn5-linA. With the aid of helper plasmid pRK600, pUT/mini-Tn5-linA was introduced into CDS-gfp, the dehydrochlorinase gene linA was integrated into the chromosome of CDS-gfp by transposing. The double labelled strain CDS-GFP-LinA was constructed. This strain was also a genetic engineering strain that was able to degrade gamma-hexachlorocyclohexane and carbofuran simultaneously. All of these results laid a foundation for the study of ecological performance of Sphingomonas sp. CDS-1.
authors.affiliation: Key Laboratory of Agricultural Environment Microbiological Engineering, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. jhxz2000cn@yahoo.com.cn
authors.forename: Jian-hong
authors.initials: JH
authors.lastname: Xu
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.forename: Qing
authors.initials: Q
authors.lastname: Hong
authors.forename: Zhi-lin
authors.initials: ZL
authors.lastname: Zhang
authors.forename: Shun-peng
authors.initials: SP
authors.lastname: Li
mesh_terms: D002235:Carbofuran / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020578:Sphingomonas / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D002235:Carbofuran