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Basic Flip Flop. Experimental Data

The most important features of the basic flip flop are the following:

• Time of the change of state

• Stability when the inducer is retired

So these are the ones that have been studied more accurately in our experiments. Overnight inoculums inducing a condition (adding 0,1 mM IPTG or growing at 42ºC) must be set the day before you want to make the experiment. The morning after a 1/100 dilution is made, and let the culture grow until de optic density reaches 0,2. In that moment the conditions are changed, to test the change between states or just retire the inducers, to observe the stability of one state in a long time.

We made the experiments in two ways:

• Qualitative, assessing that the changes actually occur. For this, we use the fluorescence microscope.

• Quantitative, with the microtiter reader we can measure how big the changes are and how quick they happen.

It’s important not to use LB media, because it contains cyclic aminoacids which make it fluorescent. For all this experiments we used Minimal Media supplemented with 0,1% casminoacids. Another problem we faced is that RFP is also excited in the wavelength we use for GFP, making our measure a little bit inaccurate. A solution to this may be insert the bistable in the bacterial chromosome, using the Mini-Tn7 based system my team-mate has developed for biobricks. The less copies of the construction, the better the regulation is, because there is more repressors for each copy of the promoters.

Several repetitions of this experiment were carried out, showing a lot of variability when the same conditions were used. We expect this great variation to be reduced in the improved constructs we are developing.

The results of one of this experiments are shown here:

The next figures show the switch from 42º to IPTG for two different experiments:

The next figures show the switch from IPTG to 42º:

As is it shown in the charts, the switch engaged by temperature is quicker and stronger than the one elicited by IPTG. Temperature changes the structure of all the cI repressor instantly, while the IPTG takes some time to enter the cell and bind to the lacI repressor.

We’ve only tried the stability of only the RFP state, finding out it last 16 h at least. This effect is very noticeable when the growth takes place in a plaque, where you can see the change of colour without UV-light.