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. 2015 Jun 10;10(6):e0119860. doi: 10.1371/journal.pone.0119860

Fig 9. Optimizing small FRET changes with inducible ligands.

Fig 9

A FRET reporter for gene expression was diversified and best sensor variants after induction of the RNA aptamer identified in bacterial colonies. (A-C) Mean ΔR/R values ± SD of cells transformed either with only the parental FRET sensor FR-Rsg1.2 [33], or a combination of the sensor with the corresponding RNA aptamer under the control of an inducible promoter following the application of tetracycline (A), doxycycline (B) or anhydrotetracycline (C) (n = 4 for each drug). ΔR/R values were calculated from FRET basal ratios at the start of the experiment and after ~3 hours. (D) An agar plate of E.coli colonies expressing different versions of the sensor imaged together with the parental sensor FR-Rsg1.2 to be optimized (negative control) and the parental sensor co-transformed with the aptamer under the control of the inducible promoter (positive control). For this purpose pieces of agar plates with these reference colonies were excised and imaged along with each screening plate (E) FRET traces of single colonies expressing mutants of the sensor in comparison to the negative and positive control. Sensors which exhibited a higher ratio change compared to FR-Rsg1.2 after 4 hours were picked and the performance was analyzed in vitro (data not shown).