Toggle Switch Achievements

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.

Figure 1: First step of cloning gel

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

Validation of the model

The fluorescent gene, put after the repressor TetR to measure its expression level, could be experimentally measure. So, the presence of fluorescence will indicate that the system is in the TetR genetic pathway and the abscence of fluorescence will indicate that the system is in the lacI pathway.

We decided to compare the model with experience as follows:

• The bacteria are first blocked in the non-fluorescent pathway (LacI).
• Then placed in a 96-well plate with at different aTc and IPTG concentration.
• Measure of the fluorescence during 10 hours.

From this experiment we get the following curve(left curve) compared to the modelling curve(right):

Figure 4: Observation of an experimental switch at two aTc concentration: 50 ng/mL and 150 ng/mL

Results predicted by simulation are the following:

Figure 5:Modelling of tetR expression for two aTc concentrations: 50 ng/mL and 150 ng/mL

Both curve were obtained with an IPTG concentration of 1mM.
We can get from the experimental graph that between an aTc concentration of 50ng/mL and 150 ng/mL there is a switch after 3 hours of experiment. Fluorescence is produced with 50ng/mL of aTc and not produced with 150ng/mL of aTc. From the modelling graph we can see that with an aTc concentration of 50ng/mL, TetR is produced and with an aTc concentration of 150ng/mL, TetR is not produced. So between these two concentrations, we observe the same switch as in the experiment. However, we see more fluorescence than expected. In fact, in the steady state no fluorescence should be observed for the red curve. But the GFP protein has an half life of 10 hours and it was impossible to get no fluorescence in a 10 hours experiment.

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. We also construct this toggle with the quorum sensing gene to get the proof of concept. And the construction with mercury repressor.

The model is validated by the experiment which shows a switch as predicted by the simulation. 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. We also construct this toggle with the quorum sensing gene to get the proof of concept.