Light-responsive control of bacterial gene expression: precise triggering of the lac promoter activity using photocaged IPTG
Light-responsive control of bacterial gene expression: precise triggering of the lac promoter activity using photocaged IPTG
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DOI：
10.1039/c4ib00027g
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发表时间：
2014-08-01
影响因子：
2.5
通讯作者：
Drepper, Thomas
中科院分区：
文献类型：
--
作者：
Binder, Dennis;Gruenberger, Alexander;Drepper, Thomas
光可以用来控制许多细胞过程，包括蛋白质功能和相互作用以及基因表达，以一种非侵入性的方式和前所未有的时空分辨率。然而，对于化学光触发器，在活细胞中从未达到紧密，渐进和均匀的光响应。在这里，我们报道了一种基于诱导剂分子异丙基- β - d -硫代半乳糖苷（IPTG）的光笼衍生物的光反应性细菌17 RNA聚合酶表达系统。对比分析了不同的大肠杆菌启动子调控表达体系在批量培养和微流控单细胞培养中的应用。lacy缺陷大肠杆菌菌株Tuner（DE3）携带额外的lad基因质粒拷贝，对IPTG浓度的增加表现出敏感和明确的反应。光笼IPTG作为合成光开关，将大肠杆菌系统转化为光遗传表达模块，允许在单细胞水平上精确和渐进地触发基因表达。
Light can be used to control numerous cellular processes including protein function and interaction as well as gene expression in a non-invasive fashion and with unprecedented spatiotemporal resolution. However, for chemical phototriggers tight, gradual, and homogeneous light response has never been attained in living cells. Here, we report on a light-responsive bacterial 17 RNA polymerase expression system based on a photocaged derivative of the inducer molecule isopropyl-beta-D-thiogalactopyranoside (IPTG). We have comparatively analyzed different Escherichia coli lac promoter-regulated expression systems in batch and microfluidic single-cell cultivation. The lacY-deficient E. coli strain Tuner(DE3) harboring additional plasmid-born copies of the lad gene exhibited a sensitive and defined response to increasing IPTG concentrations. Photocaged IPTG served as a synthetic photo-switch to convert the E. coli system into an optogenetic expression module allowing for precise and gradual light-triggering of gene expression as demonstrated at the single cell level.