Team:Paris Bettencourt/Experiments/tRNA diffusion

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Revision as of 23:52, 21 September 2011

Team IGEM Paris 2011

Characterization of the tRNA biobrick

We managed to characterize the tRNA biobrick by taking pictures under fluorescence lamp and microscopy of the strains described in the following.

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

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 glows 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.

Fluorescence lamp

A double transformation was done with 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). 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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 A double transformation was done with 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). 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.
 [[File:GFPamberwithtRNA.jpg|350px|thumb|center|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)]]
-[[File:tRNAwithoutIPTGandwith.jpg|350px|thumb|center|On the left, the non IPTG induced double transformant. On the right, the IPTG induced double transformant. The pHyperspank appears to be leaky.]]
+[[File:tRNAwithoutIPTGandwith.jpg|350px|thumb|center|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==