tBroccoli and tdBroccoli show substantially
improved performance
in bacteria compared to tSpinach2. (a) Microphotographs of bacteria
expressing tSpinach2, tBroccoli, and tdBroccoli. Respective aptamers
were expressed in E. coli and then bacterial cells
were attached to poly d-lysine coated glass-bottom dishes,
preincubated with 200 μM DFHBI-1T and imaged under the fluorescent
microscope. In these experiments, imaging was performed for 100 ms
and the brightness of the images was adjusted on the basis of the
high fluorescence signal of tdBroccoli, which results in lower signals
for the other aptamers. Cells were imaged in PBS, which lacks magnesium.
Here and in other panels, “Negative control” is the
empty vector-transformed cells. Scale bar, 2 μm. (b) Quantification
of fluorescence signal from bacterial cells in panel a, as measured
in suspension on a plate reader. Error bars indicate standard deviations
(n = 3). (c) tBroccoli, tSpinach2, and tdBroccoli
are expressed at similar levels in bacterial cells. Total RNA from
the cells from panels a and b was fractionated on urea-PAGE and stained
with DFHBI-1T and SYBR Gold. tBroccoli, tSpinach2 and tdBroccoli RNA
bands are indicated with yellow arrows. Higher molecular weight bands
are unprocessed transcripts. 5S indicated with the black arrow was
used as a loading normalization control. (d) Quantification of the
intensity of the SYBR Gold-stained bands from the panel c. Sum of
both processed and unprocessed RNA band intensity was normalized to
aptamer length. Gel image processing was performed in Image Lab 5.0
software (BioRad). Error bars indicate standard deviations (n = 3).