Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch.
Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch.
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利用强组成型启动子和茶碱激活的合成核糖开关设计枯草芽孢杆菌的诱导型基因表达系统
DOI：
10.1186/s12934-016-0599-z
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发表时间：
2016-11-22
影响因子：
6.4
通讯作者：
Zhou Z
中科院分区：
文献类型：
--
作者：
Cui W;Han L;Cheng J;Liu Z;Zhou L;Guo J;Zhou Z
背景：合成核糖开关已越来越多地用于控制和调节不同生物体中的基因表达。虽然已经开发了一套茶碱响应核糖开关的细菌，在枯草芽孢杆菌中合成核糖开关介导的全功能的表达元件很少使用，因为宿主依赖的兼容性之间的启动子和核糖开关。结果：一个新的遗传元件组成的启动子P43和茶碱核糖开关的开发和表征在B。枯草芽孢杆菌当与P43启动子（P43 '-riboE 1）组合时，茶碱-核糖开关成功地将P43的组成型表达模式转换为诱导型表达模式。茶碱可在翻译水平上激活该元件介导的表达，并具有较高的诱导率。4-mM茶碱对P43 '-riboE 1的诱导率在诱导期升高。诱导表达的水平依赖于茶碱剂量。相应地，诱导率随着茶碱剂量的增加而逐渐增加。重要的是，诱导的表达水平高于其他三种强组成型启动子，包括PsrfA，PaprE和天然P43。结果发现，表达元件内的SD序列和起始密码子之间的距离显着影响诱导表达的水平和诱导率。9 bp的间隔区适合于产生理想的表达水平和诱导率。较长的间隔物降低了活化效率。结论：所构建的茶碱诱导表达系统具有广泛的相容性和稳定性，在药物和工业蛋白的过量生产以及构建更复杂的基因电路方面具有巨大的潜力。
Background:Synthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches.Results:A novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43'-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43'-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed β-glucuronidase at equal levels, and induction ratio was similar to that of GFP.Conclusion:The constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.
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影响因子：
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通讯作者：
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