Tetracycline (TC)-sensing bioreporters using green fluorescent protein (GFP) were generated in Escherichia coli and solvent-tolerant Acinetobacter oleivorans. A TC-inducible promoter, tetH promoter, and a TetR repressor of the pAST2 plasmid recovered from sludge were used to construct plasmid-based and chromosome-based bioreporters. Two host plasmids with a broad range, pRK415 and pBBR1MCS2, and three randomly chosen chromosomal sites were used to create the reporter strains. Although the copy numbers of the two plasmids in A. oleivorans were greater than those in E. coli, GFP expression from the tetH promoter and growth under TC were significantly higher in E. coli. Thus, the E. coli bioreporter had higher GFP expression driven by TC, and the two plasmids differed in terms of their sensitivity. Our data reflected mosaic evolution of the constructed plasmids, suggesting that the plasmid replication efficiency and the tetH promoter strength differed in the two different hosts. Among the tested TC compounds, doxycycline (DC) was the most effective in promoting GFP expression. qRT-PCR data confirmed that the expression of the tetH promoter in the original pAST2 plasmid produced the most rapid response to DC. E. coli- and A. oleivorans-based plasmid reporters could detect 5 and 30 nM DC, respectively. Insertion of the GFP reporter into different positions of the A. oleivorans chromosome resulted in variations of GFP expression. Our stable A. oleivorans chromosomal bioreporter was functional in the presence of toxic organic solvents. Furthermore, the field test showed that strain A. oleivorans DR1-Tet1 could act as a sensitive bioreporter in activated sludge for DC detection.

译文

:使用绿色荧光蛋白(GFP)的四环素(TC)感应生物报告在大肠杆菌和耐溶剂不动油杆菌中产生。从污泥中回收的pAST2质粒的TC诱导型启动子,tetH启动子和TetR阻遏物用于构建基于质粒和基于染色体的生物报告基因。使用两个具有广泛范围的宿主质粒pRK415和pBBR1MCS2,以及三个随机选择的染色体位点来创建报告株。尽管在油曲霉中两个质粒的拷贝数大于在大肠杆菌中的拷贝数,但是在大肠杆菌中,来自tetH启动子的GFP表达和TC下的生长明显更高。因此,大肠杆菌生物报道基因具有较高的由TC驱动的GFP表达,并且两种质粒的敏感性不同。我们的数据反映了构建质粒的镶嵌进化,表明在两个不同宿主中质粒复制效率和tetH启动子强度不同。在测试的TC化合物中,强力霉素(DC)在促进GFP表达方面最有效。 qRT-PCR数据证实,原始pAST2质粒中tetH启动子的表达产生了对DC的最快速反应。基于大肠杆菌和油曲霉的质粒报告子分别可以检测5和30 nM DC。 GFP报告基因插入到油曲霉染色体的不同位置会导致GFP表达的变化。在有毒有机溶剂的存在下,我们稳定的油橄榄曲霉染色体生物报告剂可发挥功能。此外,现场测试表明,油曲霉DR1-Tet1菌株可作为活性污泥中的敏感生物报告物用于DC检测。

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