De novo generated toehold switch endogenous RNA sensors in vivo assays_Expt#4

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 #4 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.

本研究拟通过支点开关（toehold switch）传感器检测大肠杆菌（E. coli）小RNA RyhB，以此验证内源性RNA调控通路；该小转录本在细胞内铁水平匮乏时，可下调一系列铁相关基因的表达。
为此，将在LB培养基与100 μM硫酸亚铁中培养的、组成型表达RyhB响应型支点开关（RyhB对照组与RyhB Moirai BD1-3）的大肠杆菌细胞，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的表达水平。

结论：
当2,2'-联吡啶浓度升高至0.3 mM时达到平台期，RyhB Moirai RNA开关可最优识别RyhB小RNA。

备注：
本实验为全部4组实验中的第4组。样本标注中"bp"代表2,2'-联吡啶。本实验由Cristina Alsina博士与Gerard Minuesa博士完成，在Ivan Dotu博士的指导下于巴塞罗那科学园区的Moirai Biodesign公司开展。