De novo generated toehold switch endogenous RNA sensors in vivo assays_Expt#2

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 #2 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.