Team:Grenoble/Projet/Results

Construction of a Toggle Switch test

In order to test if our system could work, we construct a toggle switch test based on Gardner's work [1].

For realized this toggle, we used 4 primary bricks :

• pTet : BBa_R0040
• RBS-LacI-oo-pLac : BBa_Q04121
• RBS-GFP : BBa_E0240
• RBS-TetR composed of RBS BBa_B0034 and TetR BBa_C0040

First we put RBS-GFP behind RBS-TetR, and Q04121 behind pTet: both size are around 1 500 bp. The following gel shows that both constructions were at the expected size. Construction were confirmed by sequencing.

In a last step of cloning, we put RBS-tetR-RBS-GFP behind pTet-Q04121. The size is around 3 000 bp. The following gel shows that constructions was at the expected size. In addition to this test, transformation of bacteria have grown on plate with IPTG to block them in the fluorescence way. And some of the bacteria were fluorescent. Construction was also confirmed by sequencing.

Figure 2: Last step of cloning gel

Figure 3: Fluorescence test picture

To test this system, bacterias had grown in an aTc preculture to block them in a nonfluorescent way. This bacteria were put in a 96 wells plate with a two dimensionnal gradient (aTc and IPTG). After 10 hours of acquisition, we obtained the following curve :

We can get from this graph that between an aTc concentration of 50ng/mL and 150 ng/mL there is a switch after 3 hours of experiment. However, we can see more fluorescence than expected. In fact, the GFP protein has an half life of 10 hours and it was impossible to get no fluorescence.

We also did an experiment on bacterias which had grown in an IPTG preculture. But we did'nt see a switch because IPTG block bacterias in the fluorescence way. Because of the half life of GFP, it was possible to detect a switch only with bacteria which had grown with aTc.

To go further, it will be very interesting to put an LVA tag on GFP in order to control its degradation. In this case we will be abble to see the switch in both case and with more magnitude.