[Construction of double-labelled carbofuran-degrading bacterium Sphingomonas sp. CDS-1].
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.
Xu
Jian-hong
JH
Key Laboratory of Agricultural Environment Microbiological Engineering, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. jhxz2000cn@yahoo.com.cn
Wu
Jun
J
Hong
Qing
Q
Zhang
Zhi-lin
ZL
Li
Shun-peng
SP
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*
D004269:DNA, Bacterial; D002235:Carbofuran