- Prepare the working solution of poly-D-lysine freshly each time
just before it is needed. Stock solution can be stored at 4C.
Avoid multiple cycles of freeze–thaw of poly-D-lysine stock
solution. Numerous freeze-thaw cycles decrease the efficiency
of bacterial cells adhesion to the slide. - OtherE. colistrains can also be used as long as they have the
gene for T7 RNA polymerase expression, because all pET
plasmids that we use here carry a T7 promoter/terminator
system. - Perform a fresh transformation of the expression plasmids each
time to ensure efficient expression of the desired RNA
aptamers. - IPTG induction can be carried out for 2–4 h. In our experi-
ence, no significant increase in fluorescence intensity is
observed between 2-h and 4-h induction times. - To ensure proper adhesion of bacterial cells it is important to
use borosilicate glass chambers instead of the polymer based
slides. - Imaging solution must be used in live-cell imaging experiments
as it offers the following two advantages over LB medium: (1)
components of LB medium prevent adherence ofE. colito
poly-D-lysine-treated dishes, (2) live-cell imaging medium has
a lower background fluorescence than LB medium. - We observed different levels of background fluorescence for
different dyes. For example, we obtained the worst signal-to-
noise ratio with TR-DN and the best ratio with TMR-DN. - Choose the area with no cells attached carefully by using
bright-field illumination and avoid selecting attached bacterial
cells by mistake. - It is important to ensure that the dye–aptamer pairs used for
dual imaging are orthogonal to each other, i.e., they do not
bind to each other and they do not interfere with each other.
Previously, the SRB-2 aptamer was used to image RNA in live
bacterial cells using the SR-DN dye. However, the combina-
tion of the DNB–SRB-2 aptamer pair with the RG-DN
(green)–SR-DN (red) dye pair would not allow dual-color
imaging of two different RNA molecules since the DNB apta-
mer would bind to both dyes due to the presence of the same
DN contact quencher. Therefore, we conjugated another con-
tact quencher, namely, p-nitrobenzylamine (MN), to yield SR-
MN for exclusive labeling of the SRB-2 aptamer. This new
combination, SRB-2/SR-MN and DNB/TMR-DN, allowed
for simultaneous labeling of two distinct RNAs in a single live
bacterium.
302 Murat Sunbul et al.