Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I-1-I-2 regulatory elements
Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I-1-I-2 regulatory elements
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DOI：
10.1093/nar/25.6.1203
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发表时间：
1997-03-15
影响因子：
14.9
通讯作者：
Bujard, H
中科院分区：
文献类型：
--
作者：
Lutz, R;Bujard, H
以启动子活性调控参数为基础，利用lac的调控元件，构建了ara和tet操纵子转录控制序列，可独立定量调控大肠杆菌中多个基因的活性。新的启动子PLtetO-1可以在无水四环素（aTc）的5000倍范围内调节基因表达，而在IPTG和阿拉伯糖的作用下，plac1 /ara-1的活性可以控制1800倍。大肠杆菌宿主菌株从染色体定位的表达单元中产生一定数量的调节蛋白、Lac和Tet抑制因子以及AraC，提供了高度可重复性的体内条件。低丰度大肠杆菌蛋白DnaJ和限制性内切酶Cfr9I不仅表明可以实现高水平表达，而且表明在最佳抑制条件下每3代仅产生约1个mRNA，这种定量控制的潜力将为体内基因功能的研究开辟新的途径，特别是对低丰度调控基因产物的研究。该系统也将为外源基因的控制表达提供新的机会。
Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter PLtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions, Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced, This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products, The system will also provide new opportunities for the controlled expression of heterologous genes.