RNA Detection

3.2 Induction of
Reporter RNA
Expression in Bacteria

Plasmids encoding the aptamer or aptamer tagged ROI should be

transformed into an appropriate bacterial strain expressing T7 RNA

polymerase. Induction with IPTG initiates the transcription of the

RNA aptamers, which are imaged subsequently.

1. Transform BL21 Star™(DE3) competentE. colicells with
   expression plasmids (pET-tRNA, pET-SRB2, or pET-DNB)
   encoding for the respective aptamers or tRNA scaffold (con-
   trol) (seeNotes 9and 10 ).


2. Plate transformed cells on LB-agar plates supplemented with
   30 μg/mL of kanamycin for selection of the bacteria carrying
   the plasmids.


3. Pick a single colony from each plate and start overnight cul-
   tures in 5 mL of LB medium supplemented with 30μg/mL of
   kanamycin by incubating the colony at 37C with vigorous
   shaking at 150 rpm.


4. Measure the optical density (OD 600 ) for a 1:10 dilution of each
   overnight culture.


5. Start a fresh culture using the overnight culture as a starter
   culture with an OD 600 of 0.05 in a 50 mL baffled Erlenmeyer
   flask containing 10 mL of LB medium with 30 μg/μL
   kanamycin.


6. When the OD 600 reaches 0.4, the cultures are induced with
   1 mM IPTG and shaken additionally for 3 h (seeNote 11)at
   37 C.

3.3 Imaging
Aptamers in Bacteria

This section gives details about the steps for imaging various apta-

mers transcribed after the induction of pET expression vectors in

liveE. colicells.

3.3.1 Preparation of the
Poly-D-Lysine Coated Glass
Chamber Slides

Bacterial cells do not adhere to glass slides. To ensure adhesion of

the bacterial cells to a glass surface during imaging, the glass slides

must be coated with poly-D-lysine (seeNote 12).

1. 8-well Nunc™Lab-Tek™II chambered cover glass slides are
   removed from packaging and coated with 250μLof50μg/mL
   poly-D-lysine and incubated at room temperature for
   30–45 min (seeNote 8).


2. The poly-D-lysine solution is aspirated and the chamber slides
   are washed twice with 400μL sterile Millipore water to remove
   excess poly-D-lysine.


3. The excess water is aspirated after the final wash and the slides
   are allowed to air-dry at room temperature.

3.3.2 Preparation
of E. coli for Imaging

1. PrepareE. colicells expressing aptamers and tRNA scaffold
   (negative control) as described in Subheading3.2.

Visualizing RNA with Fluorogenic Aptamers in vivo 295