Team:Paris Bettencourt/Experiments/tRNA diffusion

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Team IGEM Paris 2011

Characterization of the tRNA biobrick

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:

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 / E-coli at 37°C
E-Coli at 37°C (trans image)
E-Coli at 37°C (gfp image)
E-Coli at 37°C (trans image)
E-Coli at 37°C (gfp image)
GFP amber+tRNA amber-IPTG/ E-coli at 37°C
E-Coli at 37°C (trans image)
E-Coli at 37°C (gfp image)
E-Coli at 37°C (trans image)
E-Coli at 37°C (gfp image)
GFP amber + tRNA amber + IPTG/ E-coli at 37°C
E-Coli at 37°C (trans image)
E-Coli at 37°C (gfp image)
E-Coli at 37°C (trans image)
E-Coli at 37°C (gfp image)


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