tRNA diffusion experiments

Protocol

In order to characterize the tRNA amber suppressor, as well as the pHyperspank, a special part was made: Pveg-SpoVG-GFP amber-TT. A double transformation was done with minipreps of two plasmids of different antibiotic resistances : pSB1C3 holding the tRNA under the control of pHyperspank and pSB1AK3 holding the GFP with the amber codon under the control of a constitutive promotor. Two groups of cultures of each picked colony were made. One group was cultured in rich medium without IPTG whilst the other group was cultured in rich medium with IPTG (4mM in 10mL). A culture of the strain holding GFP amber alone was grown in parallel. The two tubes of each colony were compared along with the negative control (GFP amber alone). Cultures were then diluted for microscopy, glycerols, and future experiments. We used the following strains to characterize this system:

GFP amber Strain holding GFPmut3B, mutated with a single amber mutation under the control of the constitutive promotor Pveg, in the plasmid pSB1AK3 (AmpR).

tRNA amber suppressor Strain holding the tRNA amber suppressor, under the control of IPTG inducible strong promotor pHyperspank, in the plasmid pSB1C3 (CmR).

Double transformant Double transformant of the two previous constructs in their plasmids respectively. Two groups of cultures are grown: with or without IPTG.

Direct observation under fluorescence lamp and microscopy was used to characterize this system.

Fluorescence lamp

The cultures of strains having transformed both tRNA amber suppressor and GFP amber were strongly fluorescent, while the culture of the GFP amber alone as well as the culture of the colony without IPTG induction showed basal fluorescence.

On the left, the culture of the strain holding GFP amber alone under the control of Pveg (constitutive promotor). On the right, the culture of the double transformant with IPTG induction (4mM)

On the left, the non IPTG induced double transformant. On the right, the IPTG induced double transformant. The pHyperspank shows a small quantity of leakage.

Microscopy

GFP amber

One of our strains contains only the plasmid with the GFP amber under the control of the constitutive promotor Pveg, without the amber suppressor tRNA. In this situation cells do not glow. This obervation constitutes a reliable negative control.

GFP amber with tRNA amber suppressor

The second strain contains the full construct, and the production of tRNA is tuned by pHyperspank which is activated by IPTG. In order to make a negative control, we plated them without inducing with IPTG. In this situation cells glow with a lower intensity than with the previous construct. We can also notice that some of the cells strongly leak.

GFP amber with tRNA amber induced by IPTG

The last result is given by testing the full construct with IPTG induced cells. Each cell glows with a very weak variability and the luminance is significant.