De novo generated toehold switch endogenous RNA sensors in vivo assays_Expt#3

We aimed to test endogenous RNA regulation via a toehold switch sensor to detect the E.coli small RNA RyhB, a small transcript that down-regulates a series of iron-associated genes when iron endogenous levels are low. To do so, E.coli cells constitutively expressing RyhB-responsive toehold switches (RyhB control and RyhB Moirai BD1-3) cultured in LB and 100 microM FeSO4 were induced by chelating iron with 2,2-bipyridyl at different concentrations (0 to 0.6 mM) during 1 h and the GFPmut3b-ASV expression was followed by flow cytometry (FL-1 channel).
Conclusion:
RyhB Moirai RNA switches can optimally sense ryhB sRNA in the presence of 2,2-bipyridil increasing concentrations up to 0.3 mM (plateau).
Notes:
Experiment #3 out of 4 in total. Legend from samples: 'bp' = 2,2-bibyridil. Experiments performed by Dr. Cristina Alsina and Dr. Gerard Minuesa under supervision of Dr. Ivan Dotu at Moirai Biodesign in Barcelona Scientific Park.

本研究旨在通过立足点开关传感器（toehold switch sensor）测试内源性RNA调控机制，以检测大肠杆菌（E. coli）小分子RNA RyhB——一种在细胞内铁水平较低时可下调一系列铁相关基因的小分子转录本。为此，将在LB培养基与100微摩尔硫酸亚铁中培养的、组成型表达响应RyhB的立足点开关传感器（RyhB对照株与RyhB Moirai BD1-3株）的大肠杆菌，通过不同浓度（0至0.6 mM）的2,2'-联吡啶螯合铁进行1小时诱导，并通过流式细胞术（flow cytometry，FL-1通道）监测GFPmut3b-ASV的表达情况。

结论：
RyhB Moirai RNA开关可在浓度递增至0.3 mM（平台期）的2,2'-联吡啶存在时，最优地检测RyhB小分子RNA。

备注：
本实验为全部4组实验中的第3组。样品标注说明：'bp'指代2,2'-联吡啶（原文标注为2,2-bibyridil）。本实验由Cristina Alsina博士与Gerard Minuesa博士开展，在Ivan Dotu博士的指导下于巴塞罗那科学园的Moirai Biodesign公司完成。